Polynucleotides encoding polypeptides and methods using same

The present application claims priority from U.S. Provisional Patent Application Nos. 60/607,246, filed Sep. 7, 2004, U.S. Provisional Patent Application No. 60/587,851, filed Jul. 15, 2004, and from U.S. Provisional Patent Application No. 60/539,127, filed Jan. 27, 2004, the contents of which are hereby incorporated by reference.

The present invention relates to novel secreted and non-secreted polypeptides and polynucleotides encoding same and more particularly, to therapeutic and diagnostic methods and kits utilizing same.

Extracellular proteins including receptors and their corresponding ligands play active roles in the formation, differentiation and maintenance of multicellular organisms. Any fate of an individual cell including proliferation, migration, differentiation, or interaction with other cells is typically governed by information received from distant cells and/or the immediate environment. This information is often transmitted by secreted polypeptides such as, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones, which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules are normally transferred through the cellular secretory pathway to reach their site of action at the extracellular environment.

Secreted proteins have various industrial applications, including as pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drugs available to date, including thrombolytic polypeptide sequences, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic polynucleotide or polypeptide sequences. For example, receptor immunoadhesins can be employed as therapeutic polynucleotide or polypeptide sequences to block receptor-ligand interactions. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.

Non-secreted proteins may also find application as therapeutics or diagnostics. For example, over expression of an intracellular protein (or transcript thereof) which correlates with a disease may be used to diagnose the presence of a disease or for estimating the risk of developing a disease, by the development of probes which specifically identify the over-expressed transcript or protein. In instances where the individual is at risk of suffering from a disease or other undesirable phenotype as a result of over expression of such transcript, the expression of the protein may be reduced using, for example, antisense or triple helix based strategies.

For these reasons, efforts are being made by both industry and academia to identify new, native, membrane-bound, secreted or non-secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for such proteins. Examples of such screening methods and techniques are described in, for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No. 5,536,637

The present inventors have previously designed algorithms which allow for the mass prediction of new genes and gene products and for annotating these genes and gene products [see U.S. Pat. No. 6,625,545; U.S. patent application Ser. No. 10/426,002; U.S. Patent Application No. 60/539,129 entitled Methods and systems for annotating biomolecular sequences and U.S. Patent Application No. 60/539,128 entitled METHODS OF IDENTIFYING PUTATIVE GENE PRODUCTS BY INTERSPECIES SEQUENCE COMPARISON filed concurrently herewith, assigned to the same assignee hereof and contain subject matter related, in certain respects, to the subject matter of the instant application, the teachings of all of which are incorporated herein by reference; and Example 1 of the Examples section which follows].

While applying the above-mentioned algorithms the present inventors uncovered novel naturally occurring variants of extracellular gene products, which as described above, play pivotal roles in disease onset and progression. As such these variants can be used in the diagnosis and therapy of a wide range of diseases.

According to one aspect of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 1, as determined using the LALIGN software of EMBnet Switzerland using default parameters, a nucleic acid construct comprising said isolated polynucleotide, and a host cell comprising the nucleic acid construct.

According to further features in preferred embodiments of the invention described below the nucleic acid construct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.

According to yet further features in preferred embodiments of the invention described below the nucleic acid construct further comprises positive and negative selection markers for selecting for homologous recombination events.

According to another aspect of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1, as determined using the LALIGN software of EMBnet Switzerland using default parameters or an active portion thereof.

According to yet another aspect of the present invention there is provided an antibody or an antibody fragment being capable of specifically binding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 1, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to still another aspect of the present invention there is provided an oligonucleotide specifically hybridizable with a nucleic acid sequence encoding a polypeptide having an amino acid at least 70% identical to SEQ ID NO: 1, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 1, as determined using the LALIGN software of EMBnet Switzerland using default parameters and a pharmaceutically acceptable carrier or diluent.

According to further features in preferred embodiments of the invention described below the nucleic acid sequence of the isolated polynucleotide is as set forth in SEQ ID NO: 3 or 4.

According to yet further features in preferred embodiments of the invention described below, the polypeptide encoded by said isolated polynucleotide is as set forth in SEQ ID NO: 1 or 2.

According to still another aspect of the present invention there is provided an isolated polynucleotide as set forth in SEQ ID NO: 3 or 4.

According to yet another aspect of the present invention there is provided an isolated polypeptide as set forth in SEQ ID NO: 1 or 2.

According to another aspect of the present invention there is provided a method of treating GCSF-related disease in a subject, the method comprising upregulating in the subject an expression level of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 1 as determined using the LALIGN software of EMBnet Switzerland using default parameters, thereby treating the GCSF-related disease in a subject.

According to further features in preferred embodiments of the invention described below, upregulating the expression level of the polypeptide is effected by (i) administering the polypeptide to the subject; and/or (ii) administering an expressible polynucleotide encoding the polypeptide to the subject.

According to yet further features in preferred embodiments of the invention described below the expressible polynucleotide includes a nucleic acid sequence at least 90% to SEQ ID NO:3, or is as set forth in SEQ ID NO:3.

According to another aspect of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 5, as determined using the LALIGN software of EMBnet Switzerland using default parameters, a nucleic acid construct comprising said isolated polynucleotide, and a host cell comprising the nucleic acid construct.

According to further features in preferred embodiments of the invention described below the nucleic acid construct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.

According to yet further features in preferred embodiments of the invention described below the nucleic acid construct further comprises positive and negative selection markers for selecting for homologous recombination events.

According to another aspect of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 5, as determined using the LALIGN software of EMBnet Switzerland using default parameters or an active portion thereof.

According to yet another aspect of the present invention there is provided an antibody or an antibody fragment being capable of specifically binding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 5, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to still another aspect of the present invention there is provided an oligonucleotide specifically hybridizable with a nucleic acid sequence encoding a polypeptide having an amino acid at least 70% identical to SEQ ID NO: 5, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 5, as determined using the LALIGN software of EMBnet Switzerland using default parameters and a pharmaceutically acceptable carrier or diluent.

According to further features in preferred embodiments of the invention described below the nucleic acid sequence of the isolated polynucleotide is as set forth in SEQ ID NO: 7 or 8.

According to yet further features in preferred embodiments of the invention described below, the polypeptide encoded by said isolated polynucleotide is as set forth in SEQ ID NO: 5 or 6.

According to still another aspect of the present invention there is provided an isolated polynucleotide as set forth in SEQ ID NO: 7 or 8.

According to yet another aspect of the present invention there is provided an isolated polypeptide as set forth in SEQ ID NO: 5 or 6.

According to another aspect of the present invention there is provided a method of treating TNR-3-related disease in a subject, the method comprising upregulating in the subject an expression level of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 5 as determined using the LALIGN software of EMBnet Switzerland using default parameters, thereby treating the TNR-3-related disease in a subject.

According to further features in preferred embodiments of the invention described below, upregulating the expression level of the polypeptide is effected by (i) administering the polypeptide to the subject; and/or (ii) administering an expressible polynucleotide encoding the polypeptide to the subject.

According to yet further features in preferred embodiments of the invention described below the expressible polynucleotide includes a nucleic acid sequence at least 90% identical to SEQ ID NO:7, or is as set forth in SEQ ID NO:7.

According to another aspect of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 9, as determined using the LALIGN software of EMBnet Switzerland using default parameters, a nucleic acid construct comprising said isolated polynucleotide, and a host cell comprising the nucleic acid construct.

According to further features in preferred embodiments of the invention described below the nucleic acid construct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.

According to yet further features in preferred embodiments of the invention described below the nucleic acid construct further comprises positive and negative selection markers for selecting for homologous recombination events.

According to another aspect of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 9, as determined using the LALIGN software of EMBnet Switzerland using default parameters or an active portion thereof.

According to yet another aspect of the present invention there is provided an antibody or an antibody fragment being capable of specifically binding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 9, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to still another aspect of the present invention there is provided an oligonucleotide specifically hybridizable with a nucleic acid sequence encoding a polypeptide having an amino acid at least 70% identical to SEQ ID NO: 9, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of an IL-4 polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 9, as determined using the LALIGN software of EMBnet Switzerland using default parameters and a pharmaceutically acceptable carrier or diluent.

According to further features in preferred embodiments of the invention described below the nucleic acid sequence of the isolated polynucleotide is as set forth in SEQ ID NO: 11 or 12.

According to yet further features in preferred embodiments of the invention described below, the polypeptide encoded by said isolated polynucleotide is as set forth in SEQ ID NO: 9 or 10.

According to still another aspect of the present invention there is provided an isolated polynucleotide as set forth in SEQ ID NO: 11 or 12.

According to yet another aspect of the present invention there is provided an isolated polypeptide as set forth in SEQ ID NO: 9 or 10.

According to another aspect of the present invention there is provided a method of treating IL-4-related disease in a subject, the method comprising upregulating in the subject an expression level of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 9 as determined using the LALIGN software of EMBnet Switzerland using default parameters, thereby treating the IL-4-related disease in a subject.

According to further features in preferred embodiments of the invention described below, upregulating the expression level of the polypeptide is effected by (i) administering the polypeptide to the subject; and/or (ii) administering an expressible polynucleotide encoding the polypeptide to the subject.

According to yet further features in preferred embodiments of the invention described below the expressible polynucleotide includes a nucleic acid sequence at least 90% identical to SEQ ID NO:11, or is as set forth in SEQ ID NO:11.

According to another aspect of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 13, as determined using the LALIGN software of EMBnet Switzerland using default parameters, a nucleic acid construct comprising said isolated polynucleotide, and a host cell comprising the nucleic acid construct.

According to further features in preferred embodiments of the invention described below the nucleic acid construct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.

According to yet further features in preferred embodiments of the invention described below the nucleic acid construct further comprises positive and negative selection markers for selecting for homologous recombination events.

According to another aspect of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 13, as determined using the LALIGN software of EMBnet Switzerland using default parameters or an active portion thereof.

According to yet another aspect of the present invention there is provided an antibody or an antibody fragment being capable of specifically binding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 13, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to still another aspect of the present invention there is provided an oligonucleotide specifically hybridizable with a nucleic acid sequence encoding a polypeptide having an amino acid at least 70% identical to SEQ ID NO: 13, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 13, as determined using the LALIGN software of EMBnet Switzerland using default parameters and a pharmaceutically acceptable carrier or diluent.

According to further features in preferred embodiments of the invention described below the nucleic acid sequence of the isolated polynucleotide is as set forth in SEQ ID NO: 15 or 16.

According to yet further features in preferred embodiments of the invention described below, the polypeptide encoded by said isolated polynucleotide is as set forth in SEQ ID NO: 13 or 14.

According to still another aspect of the present invention there is provided an isolated polynucleotide as set forth in SEQ ID NO: 15 or 16.

According to yet another aspect of the present invention there is provided an isolated polypeptide as set forth in SEQ ID NO: 13 or 14.

According to another aspect of the present invention there is provided a method of treating ITAV-related disease in a subject, the method comprising upregulating in the subject an expression level of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 5 as determined using the LALIGN software of EMBnet Switzerland using default parameters, thereby treating the ITAV-related disease in a subject.

According to further features in preferred embodiments of the invention described below, upregulating the expression level of the polypeptide is effected by (i) administering the polypeptide to the subject; and/or (ii) administering an expressible polynucleotide encoding the polypeptide to the subject.

According to yet further features in preferred embodiments of the invention described below the expressible polynucleotide includes a nucleic acid sequence at least 90% identical to SEQ ID NO:15, or is as set forth in SEQ ID NO:15.

According to another aspect of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 17, as determined using the LALIGN software of EMBnet Switzerland using default parameters, a nucleic acid construct comprising said isolated polynucleotide, and a host cell comprising the nucleic acid construct.

According to further features in preferred embodiments of the invention described below the nucleic acid construct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.

According to yet further features in preferred embodiments of the invention described below the nucleic acid construct further comprises positive and negative selection markers for selecting for homologous recombination events.

According to another aspect of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 17, as determined using the LALIGN software of EMBnet Switzerland using default parameters or an active portion thereof.

According to yet another aspect of the present invention there is provided an antibody or an antibody fragment being capable of specifically binding a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 17, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to still another aspect of the present invention there is provided an oligonucleotide specifically hybridizable with a nucleic acid sequence encoding a polypeptide having an amino acid at least 70% identical to SEQ ID NO: 17, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 17, as determined using the LALIGN software of EMBnet Switzerland using default parameters and a pharmaceutically acceptable carrier or diluent.

According to further features in preferred embodiments of the invention described below the nucleic acid sequence of the isolated polynucleotide is as set forth in SEQ ID NO: 19 or 20.

According to yet further features in preferred embodiments of the invention described below, the polypeptide encoded by said isolated polynucleotide is as set forth in SEQ ID NO: 17 or 18.

According to still another aspect of the present invention there is provided an isolated polynucleotide as set forth in SEQ ID NO: 19 or 20.

According to yet another aspect of the present invention there is provided an isolated polypeptide as set forth in SEQ ID NO: 17 or 18.

According to another aspect of the present invention there is provided a method of treating INR-related disease in a subject, the method comprising upregulating in the subject an expression level of a polypeptide having an amino acid sequence at least 70% identical to SEQ ID NO: 17 as determined using the LALIGN software of EMBnet Switzerland using default parameters, thereby treating the INR-related disease in a subject.

According to further features in preferred embodiments of the invention described below, upregulating the expression level of the polypeptide is effected by (i) administering the polypeptide to the subject; and/or (ii) administering an expressible polynucleotide encoding the polypeptide to the subject.

According to yet further features in preferred embodiments of the invention described below the expressible polynucleotide includes a nucleic acid sequence at least 90% identical to SEQ ID NO:19, or is as set forth in SEQ ID NO:19.

According to an additional aspect of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence at least 70% homologous to SEQ ID NO: 54, as determined using the BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, a nucleic acid construct comprising said isolated polynucleotide, and a host cell comprising the nucleic acid construct.

According to further features in preferred embodiments of the invention described below the nucleic acid construct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.

According to yet further features in preferred embodiments of the invention described below the nucleic acid construct further comprises positive and negative selection markers for selecting for homologous recombination events. The isolated polynucleotide, wherein said nucleic acid sequence is as set forth in one of SEQ ID NO: 45-53.

According to a further aspect of the present invention said polypeptide is as set forth in one of SEQ ID NO: 54-61.

According to yet an additional aspect of the present invention there is provided an isolated polynucleotide as set forth in one of SEQ ID NO: 45-53.

According to an additional aspect of the present invention there is provided an isolated polypeptide as set forth in one of SEQ ID NO: 54-61.

According to yet an additional aspect of the present invention there is provided an isolated polypeptide comprising an amino acid sequence at least 70% homologous to SEQ ID NO: 54, as determined using the BlastP software of the National Center of Biotechnology information (NCBI) using default parameters or an active portion thereof.

According to still an additional aspect of the present invention there is provided an antibody or an antibody fragment being capable of specifically binding a polypeptide having an amino acid sequence at least 70% homologous to SEQ ID NO: 54, as determined using the BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

According to yet an additional aspect of the present invention there is provided an oligonucleotide specifically hybridizable with a nucleic acid sequence encoding a polypeptide having an amino acid at least 70% homologous to SEQ ID NO: 54, as determined using the BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

According to still an additional aspect of the present invention there is provided a method of diagnosing predisposition to, or presence of cancer in a subject, the method comprising determining an expression level of a polypeptide having an amino acid at least 70% homologous to SEQ ID NO: 54, as determined using the BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, or of a polynucleotide encoding said polypeptide in a biological sample obtained from the subject, wherein said level of said polynucleotide or said level of said polypeptide is correlatable with predisposition to, or presence or absence of cancer, thereby diagnosing predisposition to, or presence of cancer in the subject.

According to further features in preferred embodiments of the invention described below the cancer is selected from the group consisting of ovarian cancer, colon cancer and lung cancer.

According to yet further features in preferred embodiments of the invention described below said determining said expression level of said polypeptide is effected via an assay selected from the group consisting of immunohistochemistry, ELISA, RIA, Western blot analysis, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.

According to further features in preferred embodiments of the invention described below said determining level of said polynucleotide is effected via an assay selected from the group consisting of PCR, RT-PCR, quantitative RT-PCR, chip hybridization, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern blot and dot blot analysis.

According to yet an additional aspect of the present invention there is provided a kit for diagnosing cancer or a predisposition thereto in a subject, the kit comprising the antibody or antibody fragment being capable of specifically binding a polypeptide having an amino acid sequence at least 70% homologous to SEQ ID NO: 54, as determined using the BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters and reagents for detecting hybridization of the antibody or antibody fragment.

According to further features in preferred embodiments of the invention described below the cancer is selected from the group consisting of ovarian cancer, colon cancer and lung cancer.

According to yet further features in preferred embodiments of the invention described below detecting hybridization of the antibody or antibody fragment is effected by an assay selected from the group consisting of immunohistochemistry, ELISA, RIA, Western blot analysis, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.

According to still further features in preferred embodiments of the invention described below said antibody or antibody fragment is coupled to an enzyme.

According to further features in preferred embodiments of the invention described below said antibody or antibody fragment is coupled to a detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety.

According to yet a further aspect of the present invention there is provided a kit for diagnosing cancer or a predisposition thereto in a subject, the kit comprising oligonucleotide specifically hybridizable with a nucleic acid sequence encoding a polypeptide having an amino acid at least 70% homologous to SEQ ID NO: 54, as determined using the BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

According to further features in preferred embodiments of the invention described below the kit further comprising reagents for detecting hybridization of the oligonucleotide.

According to yet further features in preferred embodiments of the invention described below the cancer is selected from the group consisting of ovarian cancer and lung cancer.

According to yet a further aspect of the present invention there is provided an isolated E-Selectin T1 polypeptide comprising a first amino acid sequence being at least 90% homologous to amino acids 1-176 of wild type E-SElectin corresponding to LEM2_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SKSGSCLFLHLRW (SEQ ID NO:67), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of E-Selectin T1, comprising a polypeptide having the sequence SKSGSCLFLHLRW (SEQ ID NO: 67).

According to yet a further aspect of the present invention there is provided an isolated L-Selectin T2 polypeptide comprising an amino acid sequence being at least 90% homologous to amino acids 1-254 of wild type L-Selectin corresponding to LEM1_HUMAN, contiguous to and in sequential order bridged by GE.

According to yet a further aspect of the present invention there is provided an isolated L-Selectin T3 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-317 of wild type L-Selectin corresponding to LEM1_HUMAN, bridged by S and a second amino acid sequence being at least 90% homologous to amino acids 361-372 of wild type L-Selectin corresponding to LEM1_HUMAN, wherein said first amino acid is contiguous to said bridging amino acid and said second amino acid sequence is contiguous to said bridging amino acid, and wherein said first amino acid, said bridging amino acid and said second amino acid sequence are in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for an edge portion of L-Selectin T3, comprising a first amino acid sequence being at least 90% homologous to amino acids 307-317 of wild type L-Selectin corresponding to LEM1_HUMAN, bridged by S and a second amino acid sequence being at least 90% homologous to amino acids 361-371 of wild type L-Selectin corresponding to LEM1_HUMAN, wherein said first amino acid is contiguous to said bridging amino acid and said second amino acid sequence is contiguous to said bridging amino acid, and wherein said first amino acid, said bridging amino acid and said second amino acid sequence are in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated L-Selectin T6 polypeptide consisting essentially of an amino acid sequence being at least 90% homologous to amino acids 1-316 of wild type L-Selectin corresponding to LEM1_HUMAN, contiguous to and in sequential order bridged by SE.

According to yet a further aspect of the present invention there is provided an isolated Integrin alpha M variant T8 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-288 of wild type Integrin alpha M, corresponding to ITAM_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NAALRLMLLWRVSMWIHPPFNLQILLKSK (SEQ ID NO:79), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Integrin alpha M variant T8, comprising a polypeptide having the sequence NAALRLMLLWRVSMWIHPPFNLQILLKSK (SEQ ID NO:79).

According to yet a further aspect of the present invention there is provided an isolated Integrin alpha L variant T11 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-745 of wild type Integrin alpha L, corresponding to ITAL_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRRDG (SEQ ID NO:82), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Integrin alpha L variant T11, comprising a polypeptide having the sequence VRRDG (SEQ ID NO:82).

According to yet a further aspect of the present invention there is provided an isolated Integrin alpha IIb variant T9 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-866 of wild type Integrin alpha IIb, corresponding to ITAB_HUMAN, and a second amino acid sequence being at least 90% homologous to amino acids 1020-1039 of wild type Integrin alpha IIb, corresponding to ITAB_HUMAN, wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for an edge portion of Integrin alpha IIb variant T9, comprising a first amino acid sequence being at least 90% homologous to amino acids 856-866 of wild type Integrin alpha IIb, corresponding to ITAB_HUMAN, and a second amino acid sequence being at least 90% homologous to amino acids 1020-1030 of wild type Integrin alpha IIb, corresponding to ITAB_HUMAN, wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated Integrin alpha IIb variant T8 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-981 of wild type Integrin alpha IIb, corresponding to ITAB_HUMAN, and a second amino acid sequence being at least 90% homologous to amino acids 1021-1039 of wild type Integrin alpha IIb, corresponding to ITAB_HUMAN, wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for an edge portion of Integrin alpha IIb variant T8, comprising a first amino acid sequence being at least 90% homologous to amino acids 971-981 of wild type Integrin alpha IIb, corresponding to ITAB_HUMAN, and a second amino acid sequence being at least 90% homologous to amino acids 1021-1031 of wild type Integrin alpha IIb, corresponding to ITAB_HUMAN, wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated Integrin beta-7 variant T6 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-191 of wild type Integrin beta-7, corresponding to ITB7_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence EPSAASRPVSPCLFNHCPSLCQHPGLTRAPTCPPSC (SEQ ID NO:91), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Integrin beta-7 variant T6, comprising a polypeptide having the sequence EPSAASRPVSPCLFNHCPSLCQHPGLTRAPTCPPSC (SEQ ID NO:91).

According to yet a further aspect of the present invention there is provided an isolated Interleukin 13 Receptor 1 variant T1 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-225 of wild type Interleukin 13 Receptor 1, corresponding to I131_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GPTSPYCHIGDEVST (SEQ ID NO:94), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Interleukin 13 Receptor 1variant T1, comprising a polypeptide having the sequence GPTSPYCHIGDEVST (SEQ ID NO:94).

According to yet a further aspect of the present invention there is provided an isolated Complement Component C1s variant T7 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-423 of wild type Complement Component C1s, corresponding to C1S_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GLNSDLPESSSVRWQYHCAVGCQGRGEPPQPH (SEQ ID NO:97), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Complement Component C1s variant T7, comprising a polypeptide having the sequence GLNSDLPESSSVRWQYHCAVGCQGRGEPPQPH (SEQ ID NO:97).

According to yet a further aspect of the present invention there is provided an isolated Complement Component C1s variant T8 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-65 of wild type Complement Component C1s, corresponding to C1S_HUMAN, bridged by Y and a second amino acid sequence being at least 90% homologous to amino acids 132-688 of wild type Complement Component C1s, corresponding to C1S_HUMAN, wherein said first amino acid is contiguous to said bridging amino acid and said second amino acid sequence is contiguous to said bridging amino acid, and wherein said first amino acid, said bridging amino acid and said second amino acid sequence are in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for an edge portion of Complement Component C 1 s variant T8, comprising a first amino acid sequence being at least 90% homologous to amino acids 55-65 of wild type Complement Component C1s, corresponding to C1S_HUMAN, bridged by Y and a second amino acid sequence being at least 90% homologous to amino acids 132-142 of wild type Complement Component C1s, corresponding to C1S_HUMAN, wherein said first amino acid is contiguous to said bridging amino acid and said second amino acid sequence is contiguous to said bridging amino acid, and wherein said first amino acid, said bridging amino acid and said second amino acid sequence are in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated Complement Component C5 variant T7 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-854 of wild type Complement Component C5 corresponding to CO5_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SLALSPRLECNGKISGQLQVRLPGSSDSPASASQVAGITGTHHHAQPT (SEQ ID NO:103), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Complement Component C5 variant T7, comprising a polypeptide having the sequence SLALSPRLECNGKISGQLQVRLPGSSDSPASASQVAGITGTHHHAQPT (SEQ ID NO:103).

According to yet a further aspect of the present invention there is provided an isolated Complement Component C5 variant T11 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-292 of wild type Complement Component C5 corresponding to CO5_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence RAEVR, wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Complement Component C5 variant T11, comprising a polypeptide having the sequence RAEVR.

According to yet a further aspect of the present invention there is provided an isolated Complement Receptor CR1 variant T5 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-162 of wild type Complement Component C5 corresponding to CR1_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SELKYPFLFLLPTHSNFSLE (SEQ ID NO:109), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Complement Receptor CR1 variant T5, comprising a polypeptide having the sequence SELKYPFLFLLPTHSNFSLE (SEQ ID NO:109).

According to yet a further aspect of the present invention there is provided an isolated Integrin alpha 4 variant T2 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-697 of wild type Integrin alpha 4 corresponding to ITA4_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LFHFSH (SEQ ID NO:112), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail for Integrin alpha 4 variant T2, comprising a polypeptide having the sequence LFHFSH (SEQ ID NO:112).

According to yet a further aspect of the present invention there is provided an isolated Tissue plasminogen activator (t-PA) variant T6 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-53 of wild type Tissue plasminogen activator, corresponding to TPA_HUMAN, bridged by H, and a second amino acid sequence being at least 90% homologous to amino acids 135-562 of TPA_HUMAN, wherein said first amino acid is contiguous to said bridging amino acid and said second amino acid sequence is contiguous to said bridging amino acid, and wherein said first amino acid, said bridging amino acid and said second amino acid sequence are in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for an edge portion of Tissue plasminogen activator (t-PA) variant T6, comprising a first amino acid sequence being at least 90% homologous to amino acids 43-53 of wild type Tissue plasminogen activator, corresponding to TPA_HUMAN, bridged by H and a second amino acid sequence being at least 90% homologous to amino acids 135-145 of TPA_HUMAN, wherein said first amino acid is contiguous to said bridging amino acid and said second amino acid sequence is contiguous to said bridging amino acid, and wherein said first amino acid, said bridging amino acid and said second amino acid sequence are in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated Tissue plasminogen activator (t-PA) variant T9 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-180 of wild type Tissue plasminogen activator, corresponding to TPA_HUMAN, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence TPVPRHWAWANIITAGILMGMPSPGATC (SEQ ID NO:117), wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for a tail of Tissue plasminogen activator (t-PA) variant T9, comprising a polypeptide having the sequence TPVPRHWAWANIITAGILMGMPSPGATC (SEQ ID NO: 117).

According to yet a further aspect of the present invention there is provided an isolated Thrombopoirtin variant T8 polypeptide, comprising a first amino acid sequence being at least 90% homologous to amino acids 1-132 of wild type Thrombopoietin corresponding to TPO_HUMAN, and a second amino acid sequence being at least 90% homologous to amino acids 160-353 of TPO_HUMAN, wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet a further aspect of the present invention there is provided an isolated polypeptide for an edge portion of Thrombopoirtin variant T8, comprising a first amino acid sequence being at least 90% homologous to amino acids 122-132 of TPO_HUMAN, and a second amino acid sequence being at least 90% homologous to amino acids 160-170 of TPO_HUMAN, wherein said first and said second amino acid sequences are contiguous and in a sequential order.

According to yet further aspect of the present invention there is provided an isolated chimeric polypeptide HSFLT_PEA — 1_P10, comprising a first amino acid sequence being at least 90% homologous to MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQ CRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGF YSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRV TSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHL YKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSY PDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKS VNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKD GLPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNV KPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARC DFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYIC IASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVT WILLRTVNNRTMHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNV YTGEEILQKKEITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWF KNNHKIQQEP corresponding to amino acids 1-705 of VGR1_HUMAN, which also corresponds to amino acids 1-705 of HSFLT_PEA — 1_P10, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ELYTSTSPSSSSSSPLSSSSSSSSSSSS corresponding to amino acids 706-733 of HSFLT_PEA — 1_P10, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

According to yet further aspect of the present invention there is provided and antibody or binding fragment thereof which specifically binds to the tail portion of the Vascular endothelial growth factor (VEGF) receptor HSFLT PEA-1 P10 of SEQ ID NO:533, wherein the tail portion consisting of the amino acid sequence ELYTSTSPSSSSSSPLSSSSSSSSSSSS corresponding to amino acids 706-733 of SEQ ID NO:533.

According to additional aspect of the present invention there is provided a kit comprising an antibody or binding fragment thereof which specifically binds to the tail portion of the Vascular endothelial growth factor (VEGF) receptor HSFLT PEA-1 P10 of SEQ ID NO:533, wherein the tail portion consisting of the amino acid sequence ELYTSTSPSSSSSSPLSSSSSSSSSSSS corresponding to amino acids 706-733 of SEQ ID NO:533, and at least one reagent for performing immunoassay.

According to yet further aspect of the present invention there is provided a method of detecting the binding of an antibody or binding fragment thereof which specifically binds to the tail portion of the Vascular endothelial growth factor (VEGF) receptor HSFLT PEA-1 P10 of SEQ ID NO:533, wherein the tail portion consisting of the amino acid sequence ELYTSTSPSSSSSSPLSSSSSSSSSSSS corresponding to amino acids 706-733 of SEQ ID NO:533 to a polypeptide comprising the amino acids 706-733 SEQ ID NO:533 in a sample obtained from a patient.

According to certain embodiments, the patient is suspected of having a disease selected from the group consisting of cancer, diabetes, ulcers, peripheral vascular disease and ischemia.

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a - b present the nucleic acid sequence ( FIG. 1 a ) and amino acid sequence ( FIG. 1 b ) of the GCSF variant of the present invention (SEQ ID NO:3 and 1, respectively), described in Example 2 of the Examples section which follows. The unique sequence is marked in bold and italics. The ATG and the stop codon are marked in bold and underlined.

FIG. 2 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of GCSF (transcript — 3) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as follows: =genomic DNA; =refseq mRNA; =known GenBank mRNAs; =ESTs aligned in the same orientation as their annotation; =ESTs aligned in the opposite orientation to their annotation; =ESTs without direction annotation; =predicted transcripts; =predicted polypeptide.

FIG. 3 is an amino acid sequence alignment between wild-type GCSF protein (SwissProt locus: CSF3_HUMAN; SEQ ID NO:128) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 4 is a schematic illustration showing the protein domain structure of wild-type GCSF protein (SwissProt locus: CSF3_HUMAN) and the variant of the present invention (SEQ ID NO:1). Unique region is indicated by U and arrow (SEQ ID NO:2).

FIGS. 5 a - b present the nucleic acid sequence ( FIG. 5 a ) and amino acid sequence ( FIG. 5 b ) of the TNR3 variant of the present invention (SEQ ID NOs:7 and 5, respectively), described in Example 3 of the Examples section which follows. The unique sequence is marked in bold and italics. The ATG and the stop codon are marked in bold and underlined.

FIG. 6 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of TNR3 (transcript — 19) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 7 is an amino acid sequence alignment between wild-type TNR3 protein (GenBank Accession No. P36941; TRN3_HUMAN; SEQ ID NO:129) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 8 is a schematic illustration showing the protein domain structure of wild-type TNR3 protein (SwissProt locus: TRN3_HUMAN) and the variant of the present invention (SEQ ID NO:5). Unique region is indicated by U and arrow (SEQ ID NO:6).

FIGS. 9 a - b presents the nucleic acid sequence ( FIG. 9 a ) and amino acid sequence ( FIG. 9 b ) of the IL4R variant of the present invention (Variant T5) (SEQ ID NO:11 and 9, respectively), described in Example 4 of the Examples section which follows. The unique sequence is marked in bold and italics. The ATG and the stop codon are marked in bold and underlined.

FIG. 10 is an amino acid sequence alignment between wild-type IL4R protein (SwissProt accession: IL4R_Human) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40 -dfmt=fastap.

FIG. 11 is a schematic illustration showing the protein domain structure of wild-type IL4R protein (SwissProt accession: IL4R_Human; GenBank Accession No. P24394; SEQ ID NO:130) and the variant of the present invention (SEQ ID NO:9). Unique regions are indicated by U and arrow (SEQ ID NO:10). FIGS. 12 a - b present the nucleic acid sequence (HUMVTNR_T5 (SEQ ID NO:15; FIG. 12 a ) and amino acid sequence (HUMVTNR_P5; SEQ ID NO:13, FIG. 12 b ) of the ITAV variant of the present invention (SEQ ID NOs:15 and 13, respectively), described in Example 5 of the Examples section which follows. The unique sequence is marked in bold and italics. The ATG and the stop codon are marked in bold and underlined.

FIG. 13 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of integrin α5 ITAV (Transcript — 5) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 14 is an amino acid sequence alignment between wild-type ITAV protein (SwissProt locus: ITAV_HUMAN; P06756; SEQ ID NO:131) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 15 is a schematic illustration showing the protein domain structure of wild-type ITAV protein (ITAV_HUMAN; SEQ ID NO:131) and the variant of the present invention (SEQ ID NO:13). Unique region is indicated by U (SEQ ID NO:14).

FIGS. 16 a - b present the nucleic acid sequence ( FIG. 16 a ; SEQ ID NO:19) and amino acid sequence ( FIG. 16 b ; SEQ ID NO:17) of the INR1 variant of the present invention (SEQ ID NOs:19 and 17, respectively), described in Example 6 of the Examples section which follows. The unique sequence is marked in bold and italics (SEQ ID NO:18). The ATG and the stop codon are marked in bold and underlined.

FIG. 17 is a schematic illustration depicting the viewer scheme presenting the new variant of INR1 (Transcript — 12) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 18 is an amino acid sequence alignment between wild-type INR1 protein (SwissProt locus: INR1_HUMAN; GenBank Accession No. P17181; SEQ ID NO:132) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 19 is a schematic illustration showing the protein domain structure of wild-type INR1 protein (SwissProt locus: INR1_HUMAN; SEQ ID NO:132) and the INR1 variant T12 of the present invention (SEQ ID NO:17). Unique region is indicated by U and arrow (SEQ ID NO:18).

FIG. 20 is a schematic illustration showing the protein domain structure of wild-type HGF protein (SwissProt locus: HGF_HUMAN; GenBank Accession No. P14210; SEQ ID NO:133) and the HGF variants of the present invention, as described in Example 45a-d below. The novel splice variants are as follows: Variant a: skipping exon 3; Variant b: skipping exon 4; Variant c: skipping exon 7; and Variant d: skipping exon 9.

FIG. 21 RT-PCR for identification of exon 7 skipping in Hepatocyte Growth Factor. The protocol used is described in Example 45c-2 of the Examples section which follows. Primers were taken from exon 6 (f) and 8 (r). Predicted product of full length product was 302 bp, which was found in all tissue samples. Skipping exon 7 (163 bp) was found exclusively in Colon (lane 6-arrowhead). A larger product (probably a novel exon) was found in Breast (lane 5). Tissue type cDNA pools: 1—Cervix+HeLa; 2—Uterus; 3—Ovary; 4—Placenta; 5—Breast; 6—Colon; 7—Pancreas; 8—Liver+Spleen; 9—Brain; 10—Prostate; 11—Testis; 12—Kidney; 13—Thyroid; 14—Assorted Cell-lines (5). M=1 kb ladder marker; H=H 2 O negative control.

FIG. 22 is a schematic illustration showing the protein domain structure of wild-type CART protein (SwissProt locus: CART_HUMAN; GenBank Accession No. Q16568; SEQ ID NO:134) and the CART variants of the present invention, as described in Example 46 below. Unique regions are indicated.

FIG. 23 a - b are schematic illustrations showing the protein domain structure of wild-type DPP4 protein (SwissProt locus: DPP4_HUMAN; GenBank Accession No. P27487; SEQ ID NO:135) and the DPP4 variants of the present invention, as described in Examples 47a-h below. Unique regions are indicated. The novel splice variants of DPP4 are as follows: Variant a: skipping exon 7; Variant b: skipping exon 9; Variant c: skipping exon 19; Variant d: skipping exon 21; Variant e: skipping exon 22; Variant f: skipping exon 24; and Variant g: skipping exon 25.

FIG. 24 is an illustration depicting schematic alignment of the nucleic acid sequences of wild type Troponin transcript (GenBank Accession No. NM — 003283) and variants 1, 4, 6, 9, 10, 14 and 16 (SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, and 53, respectively), as described in Example 7 below. Coding regions are shaded. Sequence region 4a codes for the unique amino acid sequence. Other regions marked in dotes code for additional novel amino acids sequences. Red arrows indicate the location of the primers and SEQ ID NOs. thereof, which were used for real-time PCR validation.

FIG. 25 is a histogram depicting the expression of troponin transcripts of the present invention in normal, benign and tumor derived ovarian samples as determined by real time PCR using a troponin-S69208_unique_region derived fragment (amplicon—SEQ ID NO:23; forward primer—SEQ ID NO:21; reverse primer—SEQ ID NO:22), as described in Example 7 of the Examples section which follows. Expression was normalized to the averaged expression of four housekeeping genes PBGD, HPRT, GAPDH and SDHA.

FIG. 26 is a histogram depicting the expression of troponin transcripts of the present invention in normal and tumor derived lung samples as determined by real time PCR using a troponin-S69208_unique_region derived fragment (amplicon—SEQ ID NO:23; forward primer—SEQ ID NO:21; reverse primer—SEQ ID NO:22), as described in Example 7 of the Examples section which follows. Expression was normalized to the averaged expression of four housekeeping genes PBGD, HPRT, Ubiquitin and SDHA.

FIG. 27 is a histogram depicting the expression of troponin transcripts for the present invention in non-cancerous, and tumor derived colon samples as determined by real time PCR using a troponin-S69208_unique_region derived fragment (SEQ ID NOs:23-amplicon), as described in Example 7c below. Expression was normalized to the averaged expression of four housekeeping genes PBGD, HPRT, RPS27A and G6PD.

FIGS. 28 a - b presents the nucleic acid sequence ( FIG. 28 a ) and amino acid sequence ( FIG. 28 b ) of the CD154 splice variant skipping exon 3 of the present invention. The novel splice variant of CD154 is described in Example 48 below. The unique sequence is marked in bold and italics. The ATG and the stop codon are marked in bold and underlined.

FIGS. 28 c - d presents the amino acid sequence ( FIG. 28 c ) and nucleic acid sequence alignment ( FIG. 28 d ) of the CD154 splice variant skipping exon 3 of the present invention and the mRNA derived from Hyper IgM syndrome (Ramesh N, et al., Int Immunol. 1993 July; 5(7): 769-73; gi AAD13982), as described in Example 48 below. Unique amino acids shared by both polypeptides are marked in bold and italics Amino acids unique for the mutated form of CD154 derived from Hyper IgM syndrome patients are marked as italics.

FIG. 28 e - f presents the nucleic acid sequence ( FIG. 28 e ) and amino acid sequence ( FIG. 280 of the CD154 splice variant skipping exon 4 of the present invention. The novel splice variant of CD154 is described in Example 48 below. The ATG and the stop codon are marked in bold and underlined.

FIG. 29 is an amino acid sequence alignment between wild-type CD154 protein (TNF5_HUMAN; GenBank Accession No. P29965; SEQ ID NO:136) and the skipping exon 3 CD154 variant of the present invention, as determined using the BlastP algorithm and default parameters. The novel splice variant of CD154 is described in Example 48 below.

FIG. 30 is an amino acid sequence alignment between wild-type CD154 protein (TNF5_HUMAN; SEQ ID NO:136) and the skipping exon 4 protein variant of the present invention, as determined using the BlastP algorithm and default parameters. The amino acids crucial for CD40 binding and for integrin α2,βIII R binding are marked. The novel splice variant of CD154 is described in Example 48 below.

FIG. 31 a presents the amino acid sequence of Macaca nemestrina CD154 protein (gi|21363028|sp|Q9BDM7|TNF5_MACNE; SEQ ID NO:137).

FIG. 31 b is an amino acid sequence alignment between wild-type CD154 protein (TNF5_HUMAN; SEQ ID NO:136) and the Macaca nemestrina CD154 protein (gi|21363028|sp|Q9BDM7|TNF5_MACNE; SEQ ID NO:137), as determined using the BlastP algorithm and default parameters. The amino acids crucial for CD40 binding and for integrin α2, βIII R binding are marked in bold.

FIG. 31 c is an amino acid sequence alignment between the CD154 skipping exon 4 splice variant of the present invention and the Macaca nemestrina CD154 protein (gi|21363028|sp|Q9BDM7|TNF5_MACNE; SEQ ID NO:137), as determined using the BlastP algorithm and default parameters. The novel splice variant of CD154 is described in Example 48 below.

FIG. 32 is a structural prediction of the partial wild type CD154 protein, demonstrating the predicted modular structure of the regions involved in CD40 binding and these involved in integrin α2,βIII binding. The prediction was performed using Cn3Dv4.1 structural viewer of NCBI.

FIG. 33 summarizes the domain structures of the variants described in Example 10.

FIG. 34 presents the domain structure of the variants described in Example 42.

FIG. 35 depicts the structure domain of the variants described in Example 49 in comparison to the known or wild-type (WT) protein.

FIG. 36 depicts the structure domain of the variants described in Example 50 in comparison to the known or wild-type (WT) protein

FIGS. 37 a - b present the nucleic acid sequence ( FIG. 37 a ) and amino acid sequence ( FIG. 37 b ) of the E-Selectin variant of the present invention (SEQ ID NO: 66 and 65, respectively), described in Example 17 below. The unique sequence is marked in bold and italics.

FIG. 38 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of E-Selectin (transcript — 1) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 39 is an amino acid sequence alignment between wild-type E-Selectin (SwissProt locus: LEM2_HUMAN; GenBank Accession No. P16581; SEQ ID NO:139) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 40 is a schematic illustration showing the protein domain structure of wild-type E-Selectin (SwissProt locus: LEM2_HUMAN; SEQ ID NO:139) and the variant of the present invention (SEQ ID NO:65). Unique region is indicated (SEQ ID NO:67).

FIGS. 41 a - b present the nucleic acid sequence ( FIG. 41 a ) and amino acid sequence ( FIG. 41 b ) of the L-Selectin variant T2 of the present invention (SEQ ID NO: 69 and 68, respectively), described in Example 33 below. The unique sequence is marked in bold and italics. The ATG and the stop codon are marked in bold and underlined.

FIGS. 41 c - d present the nucleic acid sequence ( FIG. 41 c ) and amino acid sequence ( FIG. 41 d ) of the L-Selectin variant T3 of the present invention (SEQ ID NOs:72 and 71, respectively), described in Example 33 below. The unique edge, giving rise to a unique sequence combination, is marked by H.

FIGS. 41 e - f present the nucleic acid sequence ( FIG. 41 e ) and amino acid sequence ( FIG. 410 of the L-Selectin variant T6 of the present invention (SEQ ID NOs:75 and 74, respectively), described in Example 33 below. The unique sequence is marked in bold and italics.

FIGS. 42 a - b is a schematic illustration depicting the graphical viewer scheme presenting the new variants of L-Selectin (transcripts_T2, T3 and T6) as compared to the wild type mRNA. The EST supporting the new variant T2 and T6 are indicated ( FIGS. 42 a and 42 b , respectively). Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 43 a - c is an amino acid sequence alignment between wild-type L-Selectin (SwissProt locus: LEM1_HUMAN; GenBank Accession No. P14151; SEQ ID NO:140) and the protein variants of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. FIG. 43 a is an alignment of L-selectin variant T2; FIG. 43 b is an alignment of L-selectin variant T3; FIG. 43 c is an alignment of L-selectin variant T6;

FIG. 44 is a schematic illustration showing the protein domain structure of wild-type L-Selectin (SwissProt locus: LEM1_HUMAN; SEQ ID NO:140) and the variants of the present invention (SEQ ID NOs: 68, 71 and 74). Unique region is indicated by U and arrow (SEQ ID NOs: 70, 73, and 76).

FIGS. 45 a - b present the nucleic acid sequence ( FIG. 45 a ; SEQ ID NO:78) and amino acid sequence ( FIG. 45 b ; SEQ ID NO:77) of the Integrin alpha-M variant of the present invention (integrin α-M variant Transcript_T8), described in Example 8 of the Examples section which follows. The unique sequence is marked in bold and italics. Note that there is prediction of an SNP G→C at position 2 of the transcript of the new variant (SEQ ID NO:78).

FIG. 46 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Integrin alpha-M (transcript_T8) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 47 is an amino acid sequence alignment between wild-type Integrin alpha-M (SwissProt locus: ITAM_HUMAN; GenBank Accession No. P11215; SEQ ID NO:141) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 48 is a schematic illustration showing the protein domain structure of wild-type Integrin alpha-M (SwissProt locus: ITAM_HUMAN; SEQ ID NO:141) and the variant of the present invention (SEQ ID NO:77). Unique region is indicated by U and arrow (SEQ ID NO:79).

FIGS. 49 a - b present the nucleic acid sequence ( FIG. 49 a ) and amino acid sequence ( FIG. 49 b ) of the Integrin alpha-L variant of the present invention (SEQ ID NO: 81 and 80, respectively), described in Example 18 below. The unique sequence is marked in bold and italics. The ATG and the stop codon are marked in green.

FIG. 50 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Integrin alpha-L (transcript_T11) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 51 is an amino acid sequence alignment between wild-type Integrin alpha-L (SwissProt locus: ITAL_HUMAN; GenBank Accession No. P20701; SEQ ID NO:142) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 52 is a schematic illustration showing the protein domain structure of wild-type Integrin alpha-L (SwissProt locus: ITAL_HUMAN; SEQ ID NO:142) and the variant of the present invention (SEQ ID NO:80). Unique region is indicated (SEQ ID NO: 82).

FIGS. 53 a - b present the nucleic acid sequence ( FIG. 53 a ) and amino acid sequence ( FIG. 53 b ) of the Integrin alpha-IIb variant of the present invention, transcript T9 (SEQ ID NOs:84 and 83, respectively), described in Example 28 below. The new edge, giving rise to a unique sequence combination, is marked with “][”.

FIGS. 54 a - b present the nucleic acid sequence ( FIG. 54 a ) and amino acid sequence ( FIG. 54 b ) of the Integrin alpha-IIb variant of the present invention, transcript T8 (SEQ ID NOs:87 and 86, respectively), described in Example 28 below. The new edge, giving rise to a unique sequence combination, is marked with “][”.

FIG. 55 a is an amino acid sequence alignment between wild-type Integrin alpha-IIb (SwissProt locus: ITAB_HUMAN; GenBank Accession No. P08514; SEQ ID NO:143) and the protein variant T9 of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 55 b is an amino acid sequence alignment between wild-type Integrin alpha-IIb (SwissProt locus: ITAB_HUMAN; SEQ ID NO:143) and the protein variant T8 of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 56 is a schematic illustration showing the protein domain structure of wild-type Integrin alpha-IIb (SwissProt locus: ITAB_HUMAN; SEQ ID NO:143) and the variants of the present invention (SEQ ID NOs:83 and 86).

FIGS. 57 a - b present the nucleic acid sequence ( FIG. 57 a ) and amino acid sequence ( FIG. 57 b ) of the Integrin beta-7 variant of the present invention (SEQ ID NOs:90 and 89, respectively), described in Example 20 below. The unique sequence is marked in bold and italics.

FIG. 58 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Integrin beta-7 (transcript_T6) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 59 is an amino acid sequence alignment between wild-type Integrin beta-7 (SwissProt locus: ITB7_HUMAN; GenBank Accession No. P26010; SEQ ID NO:144) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 60 is a schematic illustration showing the protein domain structure of wild-type Integrin beta-7 (SwissProt locus: ITB7_HUMAN; SEQ ID NO:144) and the variant of the present invention (SEQ ID NO:89). Unique region is indicated (SEQ ID NO:91).

FIGS. 61 a - b present the nucleic acid sequence ( FIG. 61 a ) and amino acid sequence ( FIG. 61 b ) of the Interleukin 13 receptor alpha-1 (IL-13-RA1) variant of the present invention (SEQ ID NOs:93 and 92, respectively), described in Example 31 below. The unique sequence is marked in bold and italics.

FIG. 62 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Interleukin 13 receptor alpha-1 (transcript_TD as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 63 is an amino acid sequence alignment between wild-type Interleukin 13 receptor alpha-1 (SwissProt locus: I131_HUMAN; GenBank Accession No. P78552; SEQ ID NO:145) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 64 is a schematic illustration showing the protein domain structure of wild-type Interleukin 13 receptor alpha-1 (SwissProt locus: I131_HUMAN; SEQ ID NO:145) and the variant of the present invention (SEQ ID NO:92). Unique region is indicated (SEQ ID NO:94).

FIGS. 65 a - b present the nucleic acid sequence ( FIG. 65 a ) and amino acid sequence ( FIG. 65 b ) of the Complement component C1s variant transcript T7 of the present invention (SEQ ID NOs:99 and 98, described in Example 26 below. The unique sequence is marked in bold and italics.

FIGS. 65 c - d present the nucleic acid sequence ( FIG. 65 c ) and amino acid sequence ( FIG. 65 d ) of the Complement component C1s variant transcript T8 of the present invention (SEQ ID NOs:96 and 95, respectively), described in Example 26 below. The new edge, giving rise to a unique sequence combination, is marked with “][”.

FIG. 66 is a schematic illustration depicting the graphical viewer scheme presenting the new variants of Complement component C 1 s (transcripts_T7 and T8) as compared to the wild type mRNA. The ESTs supporting the new variants are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 67 a is an amino acid sequence alignment between wild-type Complement component C1s (SwissProt locus: C1S_HUMAN; GenBank Accession No. P09871; SEQ ID NO:146) and the protein variant of the present invention, T7, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 67 b is an amino acid sequence alignment between wild-type Complement component C1s (SwissProt locus: C1S_HUMAN; SEQ ID NO:146) and the protein variant of the present invention, T8, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 68 is a schematic illustration showing the protein domain structure of wild-type Complement component C1s (SwissProt locus: C1S_HUMAN; SEQ ID NO:146) and the variants of the present invention (SEQ ID NO: 95 and 98). Unique regions are indicated (SEQ ID NO: 97 and 100).

FIGS. 69 a - b present the nucleic acid sequence ( FIG. 69 a ) and amino acid sequence ( FIG. 69 b ) of the Complement component C5 variant transcript T7 of the present invention (SEQ ID NOs:102 and 101, respectively), described in Example 21 below. The unique sequence is marked in bold and italics.

FIGS. 69 c - d present the nucleic acid sequence ( FIG. 69 c ) and amino acid sequence ( FIG. 69 d ) of the Complement component C5 variant transcript T11 of the present invention (SEQ ID NOs:105 and 104, respectively), described in Example 21 below. The unique sequence is marked in bold and italics.

FIGS. 70 a - b is a schematic illustration depicting the graphical viewer scheme presenting the new variants of Complement component C5 ( FIG. 70 a is for transcript_T7 and FIG. 70 b is for transcript T11) as compared to the wild type mRNA. The ESTs supporting the new variants are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 71 a is an amino acid sequence alignment between wild-type Complement component C5 (SwissProt locus: CO5_HUMAN; GenBank Accession No. P01031; SEQ ID NO:147) and the protein variant of the present invention, T7, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 71 b is an amino acid sequence alignment between wild-type Complement component C5 (SwissProt locus: CO5_HUMAN; SEQ ID NO:147) and the protein variant of the present invention, T11, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 72 is a schematic illustration showing the protein domain structure of wild-type Complement component C5 (SwissProt locus: CO5_HUMAN; SEQ ID NO:147) and the variants of the present invention (SEQ ID NO: 101 and 104). Unique regions are indicated (SEQ ID NO: 103 and 106).

FIGS. 73 a - b present the nucleic acid sequence ( FIG. 65 a ) and amino acid sequence ( FIG. 65 b ) of the Complement Receptor CR1 variant transcript T5 of the present invention (SEQ ID NOs:108 and 107, respectively), described in Example 25 below. The unique sequence is marked in bold and italics.

FIG. 74 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Complement Receptor CR1 (transcript_T5) as compared to the wild type mRNA. The ESTs supporting the new variants are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 75 is an amino acid sequence alignment between wild-type Complement Receptor CR1 (SwissProt locus: CR1_HUMAN; GenBank Accession No. P17927; SEQ ID NO:148) and the protein variant of the present invention, T5, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 76 is a schematic illustration showing the protein domain structure of wild-type Complement Receptor CR1 (SwissProt locus: CR1_HUMAN; SEQ ID NO:148) and the variants of the present invention (SEQ ID NO: 107). Unique regions are indicated (SEQ ID NO:109).

FIG. 77 represents the nucleic acid sequence of the Integrin alpha 4 variant of the present invention (SEQ ID NO: 111), described in Example 29 below. The unique sequence is marked in bold and italics.

FIG. 78 represents the amino acid sequence of the Integrin alpha 4 variant of the present invention (SEQ ID NO:110), described in Example 29 below. The unique sequence is marked in bold and italics.

FIG. 79 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Integrin alpha 4 (transcript — 2) as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 80 is an amino acid sequence alignment between wild-type Integrin alpha 4 (SwissProt locus: ITA4_HUMAN; GenBank Accession No. P13612; SEQ ID NO:149) and the protein variant of the present invention, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 81 is a schematic illustration showing the protein domain structure of wild-type Integrin alpha 4 (SwissProt locus: ITA4_HUMAN; SEQ ID NO:149) and the variant of the present invention (SEQ ID NO:110). Unique region is indicated (SEQ ID NO:112).

FIGS. 82 a - b present the nucleic acid sequence ( FIG. 82 a ) and amino acid sequence ( FIG. 82 b ) of the Tissue plasminogen activator (tPA) variant, transcript T6, of the present invention (SEQ ID NOs:114 and 113, respectively), described in Example 32 below. The new edge giving rise to a unique sequence junction is marked with “][”.

FIGS. 82 c - d present the nucleic acid sequence ( FIG. 82 c ) and amino acid sequence ( FIG. 82 d ) of the Tissue plasminogen activator (tPA) variant, transcript T9, of the present invention (SEQ ID NOs:116 and 115, respectively), described in Example 32 below. The unique sequence is marked in bold and italics. The new edge giving rise to a unique sequence junction is marked with “][”.

FIG. 83 a is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Tissue plasminogen activator (tPA) transcript_T6 as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 83 b is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Tissue plasminogen activator (tPA) transcript_T9 as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 84 a is an amino acid sequence alignment between wild-type Tissue plasminogen activator (tPA) (SwissProt locus: TPA_HUMAN; GenBank Accession No. P00750; SEQ ID NO:150) and the protein variant of the present invention, Tissue plasminogen activator (tPA) transcript_T6 (HUMUPAA_P4), as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 84 b is an amino acid sequence alignment between wild-type Tissue plasminogen activator (tPA) (SwissProt locus: TPA_HUMAN; SEQ ID NO:150) and the protein variant of the present invention, Tissue plasminogen activator (tPA) transcript_T9, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 85 is a schematic illustration showing the protein domain structure of wild-type Tissue plasminogen activator (tPA) (SwissProt locus: TPA_HUMAN; SEQ ID NO:150) and the variants of the present invention (SEQ ID NOs:113 and 115). Unique region is indicated (SEQ ID NO:117).

FIGS. 86 a - b present the nucleic acid sequence ( FIG. 86 a ) and amino acid sequence ( FIG. 86 b ) of the Thrombopoietin variant, transcript T8, of the present invention (SEQ ID NOs:119 and 118, respectively), described in Example 40 below. The new edge giving rise to a unique sequence junction is marked with “][”.

FIG. 87 is a schematic illustration depicting the graphical viewer scheme presenting the new variant of Thrombopoietin transcript_T8 as compared to the wild type mRNA. The ESTs supporting the new variant are indicated. Transcript indicated as “0” represents known mRNA. The pattern code is as in FIG. 2 .

FIG. 88 is an amino acid sequence alignment between wild-type

Thrombopoietin (SwissProt locus: TPO_HUMAN; GenBank Accession No. P40225; SEQ ID NO:151) and the protein variant of the present invention, Thrombopoietin transcript_T8, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 89 is a schematic illustration showing the protein domain structure of wild-type Thrombopoietin (SwissProt locus: TPO_HUMAN; SEQ ID NO:151) and the variant of the present invention (SEQ ID NO:118).

FIG. 90 is an amino acid sequence alignment between wild-type Bone morphogenetic protein receptor type II (SwissProt locus: BMR2_HUMAN; GenBank Accession No. Q13873; SEQ ID NO:152) and the protein variant of the present invention, Bone morphogenetic protein receptor type II variant (HSU20165_P5 (SEQ ID NO:120), described in Example 22, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 91 is a schematic illustration showing the protein domain structure of wild-type Bone morphogenetic protein receptor type II (SwissProt locus: BMR2_HUMAN; SEQ ID NO:152) and the variant of the present invention (SEQ ID NO:120).

FIG. 92 is an amino acid sequence alignment between wild-type Atrial natriuretic peptide receptor B (SwissProt locus: ANPB_HUMAN; GenBank Accession No. P20594; SEQ ID NO:153) and the protein variant of the present invention, Atrial natriuretic peptide receptor B variant, described in Example 35, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40 -dfmt=fastap.

FIG. 93 is a schematic illustration showing the protein domain structure of wild-type Atrial natriuretic peptide receptor B (SwissProt locus: ANPB_HUMAN; SEQ ID NO:153) and the variant of the present invention (SEQ ID NO:122).

FIG. 94 a - b is an amino acid sequence alignment between wild-type Intracellular adhesion molecule 2 (SwissProt locus: ICA2_HUMAN; GenBank Accession No. P13598; SEQ ID NO:154) and the protein variants of the present invention, Intracellular adhesion molecule 2 variant T12 ( FIGS. 94 a ) and T8 FIG. 94 b ), described in Example 41, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 95 is a schematic illustration showing the protein domain structure of wild-type Intracellular adhesion molecule 2 (SwissProt locus: ICA2_HUMAN; SEQ ID NO:154) and the variants of the present invention (SEQ ID NOs:124 and 126).

FIGS. 96 a - b present the nucleic acid sequence ( FIG. 96 a ; HSIFNABR_T14) and amino acid sequence ( FIG. 96 b ; HSIFNABR_P8) of the INR2 receptor variant, transcript T14, of the present invention (SEQ ID NOs:156 and 155, respectively), described in Example 9 of the Examples section which follows.

FIG. 97 is an amino acid sequence alignment between wild-type INR2 protein (GenBank Accession No. P48551; INR2_HUMAN; SEQ ID NO:157) and the protein variant of the present invention, HSIFNABR_P8 (SEQ ID NO:155) as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:158) in the variant of the present invention.

FIGS. 98 a - b present the nucleic acid sequence ( FIG. 98 a ; Z42185_T13) and amino acid sequence ( FIG. 98 b ; Z42185_P5) of the TR14 variant, transcript T13, of the present invention (SEQ ID NOs:159 and 160, respectively), described in Example 11 of the Examples section which follows.

FIG. 99 is an amino acid sequence alignment between wild-type TR14 protein (GenBank Accession No. Q92956; TR14_HUMAN; SEQ ID NO:161) and the protein variant of the present invention, Z42185_P5 (SEQ ID NO:160) as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:162) in the variant of the present invention.

FIGS. 100 a - b present the nucleic acid sequence ( FIG. 100 a ; HUMLAP_T18) and amino acid sequence ( FIG. 100 b ; HUMLAP_P15) of the ITB2 (integrin β2) variant, transcript T18, of the present invention (SEQ ID NOs:163 and 164, respectively), described in Example 12 of the Examples section which follows.

FIG. 101 is an amino acid sequence alignment between wild-type ITB2 protein (GenBank Accession No. P05107; ITB2_HUMAN; SEQ ID NO:165) and the protein variant of the present invention, HUMLAP_P15 (SEQ ID NO:164) as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40 -dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:166) in the variant of the present invention.

FIG. 102 is an amino acid sequence alignment between wild-type protein (ITB2_HUMAN; SEQ ID NO: 165) and the protein variant of the present invention, HUMLAP_P12 (SEQ ID NO: 168), described in Example 13 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:169) in the variant of the present invention.

FIG. 103 is an amino acid sequence alignment between wild-type protein (FC3A_HUMAN; SEQ ID NO: 173) and the protein variant of the present invention, HUMGCRFC_P3 (SEQ ID NO:171), described in Example 14 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:172) in the variant of the present invention.

FIG. 104 is an amino acid sequence alignment between wild-type protein [FC3A_HUMAN (SEQ ID NO:173)] and the protein variant of the present invention, HUMGCRFC_P4 (SEQ ID NO:175), described in Example 14 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:176) in the variant of the present invention.

FIG. 105 is an amino acid sequence alignment between wild-type protein [TNR3_HUMAN (SEQ ID NO:129)] and the protein variant of the present invention, HUMTNFRRP_P2 (SEQ ID NO:178), described in Example 15 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:179) in the variant of the present invention.

FIG. 106 is an amino acid sequence alignment between wild-type protein GCSR_HUMAN (SEQ ID NO:183) and the protein variant of the present invention, HSGCSFR2_P11 (SEQ ID NO:182), described in Example 16 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:184) in the variant of the present invention.

FIG. 107 is an amino acid sequence alignment between wild-type protein GCSR_HUMAN (SEQ ID NO:183) and the protein variant of the present invention, HSGCSFR2_P7 (SEQ ID NO:186), described in Example 16 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the absence of amino acid sequence (marked in yellow; SEQ ID NO:187) in the variant of the present invention.

FIG. 108 is an amino acid sequence alignment between wild-type protein GCSR_HUMAN (SEQ ID NO:183) and the protein variant of the present invention, HSGCSFR2_P8 (SEQ ID NO:189), described in Example 16 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in bold and italics; SEQ ID NO:190) in the variant of the present invention.

FIG. 109 is an amino acid sequence alignment between wild-type protein MI2B_HUMAN (SEQ ID NO:193) and the protein variant of the present invention, T11329_P2 (SEQ ID NO:192), described in Example 19 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:88) in the variant of the present invention.

FIG. 110 is an amino acid sequence alignment between wild-type protein VEGA_HUMAN (SEQ ID NO:196) and the protein variant of the present invention, HUMEGFAA_P6 (SEQ ID NO:195), described in Example 23 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the absence of a amino acid sequence (marked in yellow; SEQ ID NO:197) in the variant of the present invention.

FIG. 111 is an amino acid sequence alignment between wild-type protein VEGA_HUMAN (SEQ ID NO:196) and the protein variant of the present invention, HUMEGFAA_P8 (SEQ ID NO:199), described in Example 23 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the absence of amino acid sequence (marked in yellow; SEQ ID NO:200) in the variant of the present invention.

FIG. 112 is an amino acid sequence alignment between wild-type protein IL1R_HUMAN (SEQ ID NO:203) and the protein variant of the present invention, HUMIL1RA_P3 (SEQ ID NO:202), described in Example 24 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:204) in the variant of the present invention.

FIG. 113 is an amino acid sequence alignment between wild-type protein CR1_HUMAN_V4 (SEQ ID NO:260) and the protein variant of the present invention, HSCR1RS_PEA — 1_P13 (SEQ ID NO:261), described in Example 25 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 114 is an amino acid sequence alignment between wild-type protein CR1_HUMAN (SEQ ID NO:148) and the protein variant of the present invention, HSCR1RS_PEA — 1_P14 (SEQ ID NO:262), described in Example 25 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 115 is an amino acid sequence alignment between wild-type protein CR1_HUMAN (SEQ ID NO:148) and the protein variant of the present invention, HSCR1RS_PEA — 1_P15 (SEQ ID NO:263), described in Example 25 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 116 is an amino acid sequence alignment between wild-type protein CR1_HUMAN_V1 (SEQ ID NO:259) and the protein variant of the present invention, HSCR1RS_PEA — 1_P17 (SEQ ID NO:264), described in Example 25 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 117 is an amino acid sequence alignment between wild-type protein IL1B_HUMAN (SEQ ID NO:265) and the protein variant of the present invention, HSPROI1B_X1 (SEQ ID NO:270), described in Example 27 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO: 266) in the variant of the present invention.

FIG. 118 is an amino acid sequence alignment between wild-type protein PGDR_HUMAN (SEQ ID NO:267) and the protein variant of the present invention, HUMPDGFR_P6 (SEQ ID NO:272), described in Example 30 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:268) in the variant of the present invention.

FIG. 119 is an amino acid sequence alignment between wild-type protein EL3B_HUMAN (SEQ ID NO:328) and the protein variant of the present invention, HUMPRE_P4 (SEQ ID NO:274), described in Example 34 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:329) in the variant of the present invention.

FIG. 120 is an amino acid sequence alignment between wild-type protein SOMA_HUMAN (SEQ ID NO:640) and the protein variant of the present invention, HSGROW1_P11 (SEQ ID NO:276), described in Example 36 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 121 is an amino acid sequence alignment between wild-type protein (CSH_HUMAN; SEQ ID NO:330) and the protein variant of the present invention, HUMCS2_P3 (SEQ ID NO:278), described in Example 37 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:331) in the variant of the present invention.

FIG. 122 is an amino acid sequence alignment between wild-type protein (CSH_HUMAN—SEQ ID NO:330) and the protein variant of the present invention, HUMCS2_P9 (SEQ ID NO:280), described in Example 37 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:641) in the variant of the present invention.

FIG. 123 is an amino acid sequence alignment between wild-type FINC_HUMAN (SEQ ID NO:644) and the protein variant of the present invention, HUMFNC_P54 (SEQ ID NO:282), described in Example 38 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:645) in the variant of the present invention.

FIG. 124 is an amino acid sequence alignment between wild-type ITA8_HUMAN 9SEQ ID NO:327) and the protein variant of the present invention, M85929_P3 (SEQ ID NO:284), described in Example 39 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of R (marked in yellow) in the variant of the present invention.

FIG. 125 is an amino acid sequence alignment between wild-type IBP3_HUMAN (SEQ ID NO:647) and the protein variant of the present invention, S56205_P7 (SEQ ID NO:285), described in Example 43 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:659) in the variant of the present invention.

FIG. 126 is an amino acid sequence alignment between wild-type IBP3_HUMAN (SEQ ID NO:647) and the protein variant of the present invention, S56205_P15 (SEQ ID NO:287), described in Example 43 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:660) in the variant of the present invention.

FIG. 127 is an amino acid sequence alignment between wild-type RNBP_HUMAN (SEQ ID NO:648) and the protein variant of the present invention, HUMREBP_P2 (SEQ ID NO:289), described in Example 44 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:661) in the variant of the present invention.

FIG. 128 is an amino acid sequence alignment between wild-type RNBP_HUMAN (SEQ ID NO:648) and the protein variant of the present invention, HUMREBP_Skippingexon — 10_P (SEQ ID NO:291), described in Example 44 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:662) in the variant of the present invention.

FIG. 129 is an amino acid sequence alignment between wild-type RNBP_HUMAN (SEQ ID NO:648) and the protein variant of the present invention, HUMREBP_P3 (SEQ ID NO:293), described in Example 44 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:663) in the variant of the present invention.

FIG. 130 is an amino acid sequence alignment between wild-type RNBP_HUMAN (SEQ ID NO:648) and the protein variant of the present invention, HUMREBP_P4 (SEQ ID NO:295), described in Example 44 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the absence of amino acid sequence (marked in yellow) in the variant of the present invention.

FIG. 131 is an amino acid sequence alignment between wild-type RNBP_HUMAN (SEQ ID NO:648) and the protein variant of the present invention, HUMREBP_P1 (SEQ ID NO:297), described in Example 44 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:664) in the variant of the present invention.

FIG. 132 is an amino acid sequence alignment between wild-type HGF_HUMAN (SEQ ID NO:649) and the protein variant of the present invention, HSHGFR_Skipping_exon — 3_P (SEQ ID NO:299), described in Example 45 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence LH (marked in yellow;) in the variant of the present invention.

FIG. 133 is an amino acid sequence alignment between wild-type HGF_HUMAN (SEQ ID NO:649) and the protein variant of the present invention, HSHGFR_Skipping_exon — 4_P (SEQ ID NO:301), described in Example 45 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:665) in the variant of the present invention.

FIG. 134 is an amino acid sequence alignment between wild-type HGF_HUMAN (SEQ ID NO:649) and the protein variant of the present invention, HSHGFR_Skipping_exon — 7_P (SEQ ID NO:303), described in Example 45 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid S (marked in yellow) in the variant of the present invention.

FIG. 135 is an amino acid sequence alignment between wild-type HGF_HUMAN (SEQ ID NO:649) and the protein variant of the present invention, HSHGFR_Skipping_exon — 9_P (SEQ ID NO:305), described in Example 45 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:666) in the variant of the present invention.

FIG. 136 is an amino acid sequence alignment between wild-type CART_HUMAN (SEQ ID NO:650) and the protein variant of the present invention, HSU16826_Skippingexon — 2_P (SEQ ID NO:307) described in Example 46 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the absence of amino acid sequence (marked in yellow) in the variant of the present invention.

FIG. 137 is an amino acid sequence alignment between wild-type DPP4_HUMAN (SEQ ID NO:651) and the protein variant of the present invention, HSPCHDP7_Skippingexon — 7_P (SEQ ID NO:309), described in Example 47 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:667) in the variant of the present invention.

FIG. 138 is an amino acid sequence alignment between wild-type DPP4_HUMAN (SEQ ID NO:651) and the protein variant of the present invention, HSPCHDP7_Skippingexon — 9_P (SEQ ID NO:311), described in Example 47 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:668) in the variant of the present invention.

FIG. 139 is an amino acid sequence alignment between wild-type DPP4_HUMAN (SEQ ID NO:651) and the protein variant of the present invention, HSPCHDP7_Skippingexon — 19_P (SEQ ID NO:313), described in Example 47 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:669) in the variant of the present invention.

FIG. 140 is an amino acid sequence alignment between wild-type DPP4_HUMAN (SEQ ID NO:651) and the protein variant of the present invention, HSPCHDP7_Skippingexon — 21_P (SEQ ID NO:315), described in Example 47 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the presence of a unique amino acid sequence (marked in yellow; SEQ ID NO:670) in the variant of the present invention.

FIG. 141 is an amino acid sequence alignment between wild-type DPP4_HUMAN SEQ ID NO:651 and the protein variant of the present invention, HSPCHDP_Skippingexon — 22_P (SEQ ID NO:317), described in Example 47 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the unique amino acid sequence (marked in yellow; SEQ ID NO:671) in the variant of the present invention.

FIG. 142 is an amino acid sequence alignment between wild-type DPP4_HUMAN (SEQ ID NO:651) and the protein variant of the present invention, HSPCHDP7_Skippingexon — 24_P (SEQ ID NO:319), described in Example 47 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the unique amino acid sequence (marked in yellow; SEQ ID NO:672) in the variant of the present invention.

FIG. 143 is an amino acid sequence alignment between wild-type DPP4_HUMAN (SEQ ID NO:651) and the protein variant of the present invention, HSPCHDP7_Skippingexon — 25_P (SEQ ID NO:321), described in Example 47 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the unique amino acid sequence (marked in yellow; SEQ ID NO:673) in the variant of the present invention.

FIG. 144 is an amino acid sequence alignment between wild-type DPP4_HUMAN (SEQ ID NO:651) and the protein variant of the present invention, HSPCHDP7_skippingexon — 24 — 25_P (SEQ ID NO:323), described in Example 47 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap. Note the missing amino acid sequence (marked in yellow) in the variant of the present invention.

FIG. 145 is an amino acid sequence alignment between wild-type TFPI_HUMAN (SEQ ID NO:366) and the protein variant of the present invention, D12020_P5 (SEQ ID NO:367), described in Example 10 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 146 is an amino acid sequence alignment between wild-type TFPI_HUMAN (SEQ ID NO:366) and the protein variant of the present invention, D12020_P10 (SEQ ID NO:368), described in Example 10 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 147 is an amino acid sequence alignment between wild-type TFPI_HUMAN (SEQ ID NO:366) and the protein variant of the present invention, D12020_P11 (SEQ ID NO:369), described in Example 10 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 148 is an amino acid sequence alignment between wild-type EGFR_HUMAN(SEQ ID NO:427) and the protein variant of the present invention, HSEGF01_PEA — 1_P11 (SEQ ID NO:423), described in Example 42 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 149 is an amino acid sequence alignment between wild-type EGFR_HUMAN (SEQ ID NO:427) and the protein variant of the present invention, HSEGF01_PEA — 1_P14 (SEQ ID NO:424), described in Example 42 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 150 is an amino acid sequence alignment between wild-type EGFR_HUMAN (SEQ ID NO:427) and the protein variant of the present invention, HSEGF01_PEA — 1_P18 (SEQ ID NO:425), described in Example 42 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 151 is an amino acid sequence alignment between wild-type EGFR_HUMAN (SEQ ID NO:427) and the protein variant of the present invention, HSEGF01_PEA — 1_P24 (SEQ ID NO:426), described in Example 42 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 152 is an amino acid sequence alignment between wild-type VEGA_HUMAN (SEQ ID NO:196) and the protein variant of the present invention, HUMEGFAA_PEA — 2_P3 (SEQ ID NO:495), described in Example 49 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 153 is an amino acid sequence alignment between wild-type VEGA_HUMAN (SEQ ID NO:196) and the protein variant of the present invention, HUMEGFAA_PEA — 2_P14 (SEQ ID NO:496), described in Example 49 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 154 is an amino acid sequence alignment between wild-type VGR1_HUMAN (SEQ ID NO:530) and the protein variant of the present invention, HSFLT_PEA — 1_P3 (SEQ ID NO:531), described in Example 50 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 155 is an amino acid sequence alignment between wild-type VGR1_HUMAN (SEQ ID NO:530) and the protein variant of the present invention, HSFLT_PEA — 1_P4 (SEQ ID NO:532), described in Example 50 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 156 is an amino acid sequence alignment between wild-type VGR1_HUMAN (SEQ ID NO:530) and the protein variant of the present invention, HSFLT_PEA — 1_P10 (SEQ ID NO:533), described in Example 50 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 157 is an amino acid sequence alignment between wild-type VGR1_HUMAN (SEQ ID NO:530) and the protein variant of the present invention, HSFLT_PEA — 1_P12 (SEQ ID NO:534), described in Example 50 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 158 is an amino acid sequence alignment between wild-type VGR1_HUMAN (SEQ ID NO:530) and the protein variant of the present invention, HSFLT_PEA — 1_P13 (SEQ ID NO:535), described in Example 50 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 159 is an amino acid sequence alignment between wild-type VGR1_HUMAN (SEQ ID NO:530) and the protein variant of the present invention, HSFLT_PEA — 1_P14 (SEQ ID NO:536), described in Example 50 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 160 is an amino acid sequence alignment between wild-type VGR1_HUMAN (SEQ ID NO:530) and the protein variant of the present invention, HSFLT_PEA — 1_P19 (SEQ ID NO:537), described in Example 50 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 161 is an amino acid sequence alignment between wild-type VGR2_HUMAN (SEQ ID NO:555) and the protein variant of the present invention, HUMKDRZ_P8 (SEQ ID NO:556), described in Example 51 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 162 is an amino acid sequence alignment between wild-type VGR2_HUMAN (SEQ ID NO:555) and the protein variant of the present invention, HUMKDRZ_P9 (SEQ ID NO:557), described in Example 51 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 163 is an amino acid sequence alignment between wild-type CTL4_HUMAN (SEQ ID NO:564) and the protein variant of the present invention, HUMCTLA4B_PEA — 1_P3 (SEQ ID NO:565), described in Example 52 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 164 is an amino acid sequence alignment between wild-type TR1A_HUMAN (SEQ ID NO:631) and the protein variant of the present invention, HSTNFR1A_PEA — 1_P11 (SEQ ID NO:632), described in Example 53 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 165 is an amino acid sequence alignment between wild-type TR1A_HUMAN (SEQ ID NO:631) and the protein variant of the present invention, HSTNFR1A_PEA — 1_P15 (SEQ ID NO:633), described in Example 53 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 166 is an amino acid sequence alignment between wild-type TR1A_HUMAN (SEQ ID NO:631) and the protein variant of the present invention, HSTNFR1A_PEA — 1_P19 (SEQ ID NO:634), described in Example 53 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 167 is an amino acid sequence alignment between wild-type TR1A_HUMAN (SEQ ID NO:631) and the protein variant of the present invention, HSTNFR1A_PEA — 1_P20 (SEQ ID NO:635), described in Example 53 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 168 is an amino acid sequence alignment between wild-type TR1A_HUMAN (SEQ ID NO:631) and the protein variant of the present invention, HSTNFR1A_PEA — 1_P22 (SEQ ID NO:636), described in Example 53 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 169 is an amino acid sequence alignment between wild-type TR1A_HUMAN (SEQ ID NO:631) and the protein variant of the present invention, HSTNFR1A_PEA — 1_P23 (SEQ ID NO:637), described in Example 53 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 170 is an amino acid sequence alignment between wild-type TR1A_HUMAN (SEQ ID NO:631) and the protein variant of the present invention, HSTNFR1A_PEA — 1_P24 (SEQ ID NO:638), described in Example 53 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 171 is an amino acid sequence alignment between wild-type TR1A_HUMAN (SEQ ID NO:631) and the protein variant of the present invention, HSTNFR1A_PEA — 1_P28 (SEQ ID NO:639), described in Example 53 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 172 is an amino acid sequence alignment between wild-type CO5_HUMAN_V1 (SEQ ID NO:730) and the protein variant of the present invention, HUMC5_PEA — 3_P12 (SEQ ID NO:727, described in Example 54 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 173 is an amino acid sequence alignment between wild-type CO5_HUMAN (SEQ ID NO:730) and the protein variant of the present invention, HUMC5_PEA — 3_P13 (SEQ ID NO:728), described in Example 54 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 174 is an amino acid sequence alignment between wild-type CO5_HUMAN (SEQ ID NO:730) and the protein variant of the present invention, HUMC5_PEA — 3_P15 (SEQ ID NO:729), described in Example 54 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 175 is an amino acid sequence alignment between wild-type FA8_HUMAN (SEQ ID NO:769) and the protein variant of the present invention, HUMFVIII_PEA — 1_P9 (SEQ ID NO:765), described in Example 55 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 176 is an amino acid sequence alignment between wild-type FA8_HUMAN (SEQ ID NO:769) and the protein variant of the present invention, HUMFVIII_PEA — 1_P10 (SEQ ID NO:766), described in Example 55 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 177 is an amino acid sequence alignment between wild-type FA8_HUMAN (SEQ ID NO:768) and the protein variant of the present invention, HUMFVIII_PEA — 1_P11 (SEQ ID NO:767), described in Example 55 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 178 is an amino acid sequence alignment between wild-type FA8_HUMAN (SEQ ID NO:769) and the protein variant of the present invention, HUMFVIII_PEA — 1_P13 (SEQ ID NO:768), described in Example 55 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 179 is an amino acid sequence alignment between wild-type C1S_HUMAN (SEQ ID NO:821) and the protein variant of the present invention, HUMC1RS_PEA — 1_P8 (SEQ ID NO:816), described in Example 56 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 180 is an amino acid sequence alignment between wild-type C1S_HUMAN (SEQ ID NO:821) and the protein variant of the present invention, HUMC1RS_PEA — 1_P21 (SEQ ID NO:817), described in Example 56 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 181 is an amino acid sequence alignment between wild-type C1S_HUMAN (SEQ ID NO:821) and the protein variant of the present invention, HUMC1RS_PEA — 1_P22 (SEQ ID NO:818), described in Example 56 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 182 is an amino acid sequence alignment between wild-type C1S_HUMAN (SEQ ID NO:821) and the protein variant of the present invention, HUMC1RS_PEA — 1_P23 (SEQ ID NO:819), described in Example 56 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 183 is an amino acid sequence alignment between wild-type C1S_HUMAN (SEQ ID NO:821) and the protein variant of the present invention, HUMC1RS_PEA — 1_P24 (SEQ ID NO:820), described in Example 56 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 184 is an amino acid sequence alignment between wild-type SOMA_HUMAN (SEQ ID NO:850) and the protein variant of the present invention, HSGROW1_PEA — 1_P7 (SEQ ID NO:851), described in Example 57 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 185 is an amino acid sequence alignment between wild-type SOMA_HUMAN (SEQ ID NO:850) and the protein variant of the present invention, HSGROW1_PEA — 1_P11 (SEQ ID NO:852), described in Example 57 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 186 is an amino acid sequence alignment between wild-type SOMA_HUMAN (SEQ ID NO:850) and the protein variant of the present invention, HSGROW1_PEA — 1_P12 (SEQ ID NO:853), described in Example 57 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 187 is an amino acid sequence alignment between wild-type SOMA_HUMAN (SEQ ID NO:850) and the protein variant of the present invention, HSGROW1_PEA — 1_P18 (SEQ ID NO:854), described in Example 57 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 188 is an amino acid sequence alignment between wild-type SOMA_HUMAN (SEQ ID NO:850) and the protein variant of the present invention, HSGROW1_PEA — 1_P21 (SEQ ID NO:855), described in Example 57 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 189 depicts the variants domain structure in comparison to the known or wild-type (WT) protein. T11 contains the convertase binding site on the β-chain (and maybe other, not yet know, binding sites), and thus, might interfere with the binding of the convertase with the WT C5, and might serve as an antagonist. T16, T14 might compete with C5 on its interaction with C5 convertase, and may thus serve as an antagonist of complement activation.

FIG. 190 depicts factor VIII launched products.

FIG. 191 depicts factor VIII related development.

FIG. 192 depicts factor VIII clinical and preclinical developments.

FIG. 193 depicts the domain structure of the variants described in Example 55 in comparison to the known or wild-type (WT) protein (Factor VIII).

FIG. 194 depicts the Complement Pathway, described in Example 56 of the Examples section which follows.

FIG. 195 depicts C5 clinical developments, described in Example 56 of the Examples section which follows.

FIG. 196 depicts C5 preclinical developments, described in Example 56 of the Examples section which follows.

FIG. 197 depicts CR1 clinical developments, described in Example 56 of the Examples section which follows.

FIGS. 198 a - b depict the C1s clinical development ( FIG. 198 a ) and related drugs ( FIG. 198 b ), described in Example 56 of the Examples section which follows.

FIG. 199 depict the domain structure of the variants described in Example 56 compared to WT. P455 is predicted to be bound and cleaved by C1r, and to bind but not cleave, C4. P292, P330 and P405 are predicted to interact with C1r and to act as dominant negative. P621 will not be bound and cleaved by C1r, thus will not get activated however, it is predicted to retain its ability to bind C4 and thus, might serve as an antagonist.

FIG. 200 depicts GH antagonists-launched products, described in Example 57 of the Examples section which follows.

FIG. 201 depicts GH antagonists-clinical development, described in Example 57 of the Examples section which follows.

FIG. 202 depicts the domain structure of the variants described in Example 57 of the Examples section which follows, in comparison to WT.

FIG. 203 depicts the domain structure of the variants described in Example 51 of the Examples section which follows, in comparison to WT.

FIG. 204 depicts the domain structure of the variants described in Example 53 of the Examples section which follows, in comparison to WT.

FIG. 205 is an amino acid sequence alignment between wild-type PSPD_HUMAN (SEQ ID NO:858) and the protein variant of the present invention, D45608_P3 (SEQ ID NO:857), described in Example 58 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap. Note the amino acid sequence which is missing in the D45608_P3 variant (marked in yellow).

FIG. 206 is an amino acid sequence alignment between wild-type TR1B_HUMAN (SEQ ID NO:862) and the protein variant of the present invention, HUMTNFRII_PEA — 1_P7 (SEQ ID NO:696), described in Example 59 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 207 is an amino acid sequence alignment between wild-type TR1B_HUMAN (SEQ ID NO:862) and the protein variant of the present invention, HUMTNFRII_PEA — 1_P15 (SEQ ID NO:697), described in Example 59 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 208 is an amino acid sequence alignment between wild-type TR1B_HUMAN (SEQ ID NO:862) and the protein variant of the present invention, HUMTNFRII_PEA — 1_P17 (SEQ ID NO:699), described in Example 59 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 209 is an amino acid sequence alignment between wild-type TR1B_HUMAN (SEQ ID NO:862) and the protein variant of the present invention, HUMTNFRII_PEA — 1_P18 (SEQ ID NO:860), described in Example 59 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 210 is an amino acid sequence alignment between wild-type TR1B_HUMAN (SEQ ID NO:862) and the protein variant of the present invention, HUMTNFRII_PEA — 1_P19 (SEQ ID NO:861), described in Example 59 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 211 depicts the domain structure of TNFRII variants in comparison to the known or wild-type (WT) protein, described in Example 59.

FIG. 212 depicts the clinical trials involve TNR3 lymphotoxin beta.

FIG. 213 is an amino acid sequence alignment between wild-type TNR3_HUMAN (SEQ ID NO:129) and the protein variant of the present invention, HUMTNFRRP_P2 (SEQ ID NO:898), described in Example 60 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 214 is an amino acid sequence alignment between wild-type TNR3_HUMAN (SEQ ID NO:129) and the protein variant of the present invention, HUMTNFRRP_P4 (SEQ ID NO:899), described in Example 60 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 215 is an amino acid sequence alignment between wild-type TNR3_HUMAN (SEQ ID NO:129) and the protein variant of the present invention, HUMTNFRRP_P9 (SEQ ID NO:900), described in Example 60 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 216 depicts the domain structure of the variants described in Example 60 of the Examples section which follows in comparison to WT TNR3_HUMAN (SEQ ID NO:129)

FIG. 217 depicts the IL-12 clinical developments.

FIG. 218 is an amino acid sequence alignment between wild-type I12A_HUMAN (SEQ ID NO:933) and the protein variant of the present invention, HUMCLMF35_PEA — 1_PEA — 2_P14 (SEQ ID NO:927), described in Example 61 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 219 is an amino acid sequence alignment between wild-type I12A_HUMAN (SEQ ID NO:933) and the protein variant of the present invention, HUMCLMF35_PEA — 1_PEA — 2_P15 (SEQ ID NO:928), described in Example 61 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 220 is an amino acid sequence alignment between wild-type I12A_HUMAN (SEQ ID NO:933) and the protein variant of the present invention, HUMCLMF35_PEA — 1_PEA — 2_P16 (SEQ ID NO:929), described in Example 61 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 221 is an amino acid sequence alignment between wild-type I12A_HUMAN (SEQ ID NO:933) and the protein variant of the present invention, HUMCLMF35_PEA — 1_PEA — 2_P17 (SEQ ID NO:930), described in Example 61 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 222 is an amino acid sequence alignment between wild-type I12A_HUMAN (SEQ ID NO:933) and the protein variant of the present invention, HUMCLMF35_PEA — 1_PEA — 2_P20 (SEQ ID NO:931), described in Example 61 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 223 is an amino acid sequence alignment between wild-type I12A_HUMAN (SEQ ID NO:933) and the protein variant of the present invention, HUMCLMF35_PEA — 1_PEA — 2_P22 (SEQ ID NO:932), described in Example 61 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 224 depicts the domain structure of the variants described in Example 61 of the Example section which follows in comparison with the WT IL12.

FIG. 225 depicts the IL6 clinical developments.

FIG. 226 depicts the domain structure of the variants described in Example 62 of the Examples section which follows in comparison to WT IL6.

FIG. 227 is an amino acid sequence alignment between wild-type IL6_HUMAN (SEQ ID NO:959) and the protein variant of the present invention, S56892_PEA — 1_PEA — 1_P8 (SEQ ID NO:956), described in Example 62 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 228 is an amino acid sequence alignment between wild-type IL6_HUMAN (SEQ ID NO:959) and the protein variant of the present invention, S56892_PEA — 1_PEA — 1_P9 (SEQ ID NO:957), described in Example 62 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 229 is an amino acid sequence alignment between wild-type IL6_HUMAN (SEQ ID NO:959) and the protein variant of the present invention, S56892_PEA — 1_PEA — 1_P11 (SEQ ID NO:958), described in Example 62 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 230 is an amino acid sequence alignment between wild-type TGR2_HUMAN (SEQ ID NO:974) and the protein variant of the present invention, HUMTGFBIIR_PEA — 1_P9 (SEQ ID NO:972), described in Example 63 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 231 is an amino acid sequence alignment between wild-type TGR2_HUMAN (SEQ ID NO:974) and the protein variant of the present invention, HUMTGFBIIR_PEA — 1_P14 (SEQ ID NO:973), described in Example 63 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 232 depicts the domain structure of the variants described in Example 63 of the Examples section which follows in comparison to TGR2_HUMAN variant.

FIG. 233 depicts GCSF launched products, described in Example 64 of the Examples section which follows.

FIG. 234 depicts GCSF clinical developments described in Example 64 of the Examples section which follows.

FIG. 235 depicts GCSF Preclinical developments described in Example 64 of the Examples section which follows.

FIG. 236 depicts GCSF domain structure of the variants described in Example 64 of the Examples section which follows, in comparison to WT GCSF.

FIG. 237 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P5 (SEQ ID NO:1000), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 238 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P6 (SEQ ID NO:1001), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 239 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P7 (SEQ ID NO:1002), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 240 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P8 (SEQ ID NO:1003), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 241 is an amino acid sequence alignment between wild-type Q8N4W3 (SEQ ID NO:1012) and the protein variant of the present invention, HUMGCSF_PEA — 1_P8 (SEQ ID NO:1003), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 242 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P9 (SEQ ID NO:1004), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 243 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P13 (SEQ ID NO:1005), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 244 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P14 (SEQ ID NO:1006), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 245 is an amino acid sequence alignment between Q8N4W3 (SEQ ID NO:1012) and the protein variant of the present invention, HUMGCSF_PEA — 1_P14 (SEQ ID NO:1006), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40 -dfmt=fastap.

FIG. 246 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P16 (SEQ ID NO:1007), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 247 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P18 (SEQ ID NO:1008), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 248 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P19 (SEQ ID NO:1009), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 249 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P20 (SEQ ID NO:1010), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 250 is an amino acid sequence alignment between Q8N4W3 (SEQ ID NO:1012) and the protein variant of the present invention, HUMGCSF_PEA — 1_P20 (SEQ ID NO:1010), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40 -dfmt=fastap.

FIG. 251 is an amino acid sequence alignment between wild-type CSF3_HUMAN (SEQ ID NO:128) and the protein variant of the present invention, HUMGCSF_PEA — 1_P21 (SEQ ID NO:1011), described in Example 64 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 252 depicts the TGF beta clinical studies described in Example 65 of the Examples section which follows.

FIG. 253 depicts TGF beta preclinical studies described in Example 65 of the Examples section which follows.

FIG. 254 depicts domain structure of the variants described in Example 65 of the Examples section which follows in comparison to WT TGF-beta.

FIG. 255 is an amino acid sequence alignment between wild-type TGF1_HUMAN (SEQ ID NO:1048) and the protein variant of the present invention, HSTGFB1_PEA — 1_P2 (SEQ ID NO:1043), described in Example 65 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 256 is an amino acid sequence alignment between wild-type TGF1_HUMAN (SEQ ID NO:1048) and the protein variant of the present invention, HSTGFB1_PEA — 1_P3 (SEQ ID NO:1044), described in Example 65 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 257 is an amino acid sequence alignment between wild-type TGF1_HUMAN (SEQ ID NO:1048) and the protein variant of the present invention, HSTGFB1_PEA — 1_P5 (SEQ ID NO:1045), described in Example 65 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 258 is an amino acid sequence alignment between wild-type TGF1_HUMAN (SEQ ID NO:1048) and the protein variant of the present invention, HSTGFB1_PEA — 1_P7 (SEQ ID NO:1046), described in Example 65 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 259 is an amino acid sequence alignment between wild-type TGF1_HUMAN (SEQ ID NO:1048) and the protein variant of the present invention, HSTGFB1_PEA — 1_P10 (SEQ ID NO:1047), described in Example 65 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 260 is an amino acid sequence alignment between wild-type TPA_HUMAN (SEQ ID NO:150) and the protein variant of the present invention, HUMUPAA_P14 (SEQ ID NO:1102), described in Example 66 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 261 is an amino acid sequence alignment between wild-type TPA_HUMAN (SEQ ID NO:150) and the protein variant of the present invention, HUMUPAA_P17 (SEQ ID NO:1103), described in Example 66 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 262 is an amino acid sequence alignment between wild-type TPA_HUMAN (SEQ ID NO:150) and the protein variant of the present invention, HUMUPAA_P20 (SEQ ID NO:1104), described in Example 66 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 263 depicts the domain structure of the variants described in Example 66 of the Examples section which follows in comparison to the WT TPA_HUMAN.

FIG. 264 is an amino acid sequence alignment between wild-type DRN1_HUMAN (SEQ ID NO:1131) and the protein variant of the present invention, HUMDNASEI_PEA — 1_P3 (SEQ ID NO:1127), described in Example 67 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 265 is an amino acid sequence alignment between wild-type DRN1_HUMAN (SEQ ID NO:1131) and the protein variant of the present invention, HUMDNASEI_PEA — 1_P4 (SEQ ID NO:1128), described in Example 67 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 266 is an amino acid sequence alignment between wild-type DRN1_HUMAN (SEQ ID NO:1131) and the protein variant of the present invention, HUMDNASEI_PEA — 1_P6 (SEQ ID NO:1129), described in Example 67 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 267 is an amino acid sequence alignment between wild-type DRN1_HUMAN (SEQ ID NO:1131) and the protein variant of the present invention, HUMDNASEI_PEA — 1_P10 (SEQ ID NO:1130), described in Example 67 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 268 is an amino acid sequence alignment between wild-type TNFA_HUMAN (SEQ ID NO:1155) and the protein variant of the present invention, HUMTNFAA_PEA — 1_P6 (SEQ ID NO:1144), described in Example 68 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 269 is an amino acid sequence alignment between wild-type TNFA_HUMAN (SEQ ID NO:1155) and the protein variant of the present invention, HUMTNFAA_PEA — 1_P7 (SEQ ID NO:1145), described in Example 68 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 270 is an amino acid sequence alignment between wild-type TNFA_HUMAN_V1 (SEQ ID NO:1147) and the protein variant of the present invention, HUMTNFAA_PEA — 1_P8 (SEQ ID NO:1146), described in Example 68 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0 -matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 271 is an amino acid sequence alignment between wild-type MEC2_HUMAN (SEQ ID NO:1154) and the protein variant of the present invention, M62144_P3 SEQ ID NO:1148), described in Example 69 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 272 is an amino acid sequence alignment between wild-type MEC2_HUMAN (SEQ ID NO:1154) and the protein variant of the present invention, M62144_P2 (SEQ ID NO:1150), described in Example 69 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

FIG. 273 is an amino acid sequence alignment between wild-type MEC2_HUMAN (SEQ ID NO:1154) and the protein variant of the present invention, M62144_P4 (SEQ ID NO:1152), described in Example 69 of the Examples section which follows, as determined using the Smith&Waterman model query db, with the following parameters: -mode=qglobal -onestrand -gapext=0-matrix=identity -out=g -gapop=40-dfmt=fastap.

The present invention is of novel secreted and non-secreted polypeptides and polynucleotides encoding same, which can be used for the diagnosis and treatment of a wide range of diseases, such as cancer and inflammatory diseases.

According to still other preferred embodiments, the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein, including any oligopeptide or peptide relating to such an amino acid sequence or fragment, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges. The present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.

The present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.

In another embodiment, the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides. Such bridges, tails, heads and/or insertions are described in greater detail below with regard to the Examples.

As used herein a “tail” refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.

As used herein a “head” refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein.

As used herein “an edge portion” refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein. An edge may optionally arise due to a join between the above “known protein” portion of a variant and the tail, for example, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein. A “bridge” may optionally be an edge portion as described above, but may also include a join between a head and a “known protein” portion of a variant, or a join between a tail and a “known protein” portion of a variant, or a join between an insertion and a “known protein” portion of a variant.

In another embodiment, this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention. Preferably such antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).

In another embodiment, this invention provides an isolated nucleic acid molecule encoding for a splice variant according to the present invention, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention. In another embodiment, this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.

Nucleic Acid Sequences and Oligonucleotides

Various embodiments of the present invention encompass nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or artificially induced, either randomly or in a targeted fashion.

The present invention encompasses nucleic acid sequences described herein; fragments thereof, sequences hybridizable therewith, sequences homologous thereto [e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95% or more say 100% identical to the nucleic acid sequences set forth below], sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion. The present invention also encompasses homologous nucleic acid sequences (i.e., which form a part of a polynucleotide sequence of the present invention) which include sequence regions unique to the polynucleotides of the present invention.

In cases where the polynucleotide sequences of the present invention encode previously unidentified polypeptides, the present invention also encompasses novel polypeptides or portions thereof, which are encoded by the isolated polynucleotide and respective nucleic acid fragments thereof described hereinabove.

Thus, the present invention provides isolated polynucleotides each encoding a polypeptide which is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, %, at least 85%, %, at least 90%, at least 95% or more, say 100% identical to a polypeptide sequence listed in the Examples section or sequence listing, as determined using the LALIGN software of EMBnet Switzerland using default parameters.

A “nucleic acid fragment” or an “oligonucleotide” or a “polynucleotide” are used herein interchangeably to refer to a polymer of nucleic acids. A polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers to a sequence, which is composed of genomic and cDNA sequences. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.

Preferred embodiments of the present invention encompass oligonucleotide probes.

An example of an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).

Alternatively, an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).

Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988) and “Oligonucleotide Synthesis” Gait, M. J., ed. (1984) utilizing solid phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting and purification by for example, an automated trityl-on method or HPLC.

Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases. Preferably, the oligonucleotide of the present invention features at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the biomarkers of the present invention.

The oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3′ to 5′ phosphodiester linkage.

Preferably used oligonucleotides are those modified at one or more of the backbone, internucleoside linkages or bases, as is broadly described hereinunder.

Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat. Nos. 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466, 677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms can also be used.

Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example for such an oligonucleotide mimetic, includes peptide nucleic acid (PNA). United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, “unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further bases particularly useful for increasing the binding affinity of the oligomeric compounds of the invention include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, as disclosed in U.S. Pat. No. 6,303,374.

It is not necessary for all positions in a given oligonucleotide molecule to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

It will be appreciated that oligonucleotides of the present invention may include further modifications for more efficient use as diagnostic agents and/or to increase bioavailability, therapeutic efficacy and reduce cytotoxicity.

Expression of the Polynucleotide Sequence of the Present Invention

To enable cellular expression of the polynucleotides of the present invention, a nucleic acid construct (or an “expression vector”) according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element. As used herein, the phrase “cis acting regulatory element” refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.

Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed. Examples of cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). The nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.

Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40.

In addition to the elements already described, the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

The expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

The nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication. Preferably, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/−), pGL3, PzeoSV2 (+/−), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. Examples of retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif., includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the transgene is transcribed from CMV promoter. Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5′LTR promoter.

Viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by the present invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein. For example, bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) as described in Liang C Y et al., 2004 (Arch Virol. 149: 51-60).

Recombinant viral vectors are useful for in vivo expression of the polynucleotide sequence of the present invention (e.g., SEQ ID NO: 3, 7, 11, 15, 19, or 45) since they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

Various methods can be used to introduce the expression vector of the present invention into stem cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.

Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of the present invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide. For example, the expression of a fusion protein or a cleavable fusion protein comprising Met variant of the present invention and a heterologous protein can be engineered. Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein. Where a cleavage site is engineered between the Met moiety and the heterologous protein, the Met moiety can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-158591.

As mentioned hereinabove, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence. Mammalian expression systems can also be used to express the polypeptides of the present invention.

Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).

In yeast, a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. No. 5,932,447. Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.

In cases where plant expression vectors are used, the expression of the coding sequence can be driven by a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter to TMV [Takamatsu et al. (1987) EMBO J. 6:307-311] can be used. Alternatively, plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al. (1986) Mol. Cell. Biol. 6:559-565] can be used. These constructs can be introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

Other expression systems such as insects and mammalian host cell systems which are well known in the art and are further described hereinbelow can also be used by the present invention.

Recovery of the recombinant polypeptide is effected following an appropriate time in culture. The phrase “recovering the recombinant polypeptide” refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. Not withstanding the above, polypeptides of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

Hybridization Assays

Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non-limiting examples of probes according to the present invention were previously described). Traditional hybridization assays include PCR, RT-PCR, Real-time PCR, RNase protection, in-situ hybridization, primer extension, Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection) (NAT type assays are described in greater detail below). More recently, PNAs have been described (Nielsen et al. 1999, Current Opin. Biotechnol. 10:71-75). Other detection methods include kits containing probes on a dipstick setup and the like.

Hybridization based assays which allow the detection of a variant of interest (i.e., DNA or RNA) in a biological sample rely on the use of oligonucleotides which can be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides long.

Thus, the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.

Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10% dextrane sulfate, 1 M NaCl, 1% SDS and 5×106 cpm 32P labeled probe, at 65° C., with a final wash solution of 0.2×SSC and 0.1% SDS and final wash at 65° C. and whereas moderate hybridization is effected using a hybridization solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDS and 5×106 cpm 32P labeled probe, at 65° C., with a final wash solution of 1×SSC and 0.1% SDS and final wash at 50° C.

More generally, hybridization of short nucleic acids (below 200 bp in length, e.g. 17-40 bp in length) can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency; (i) hybridization solution of 6×SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 mg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 1-1.5° C. below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm; (ii) hybridization solution of 6×SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 mg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 2-2.5° C. below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm, final wash solution of 6×SSC, and final wash at 22° C.; (iii) hybridization solution of 6×SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 mg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature.

The detection of hybrid duplexes can be carried out by a number of methods. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Such labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. A label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.

Probes can be labeled according to numerous well known methods. Non-limiting examples of radioactive labels include 3H, 14C, 32P, and 35S, Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radio-nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.

For example, oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent. Alternatively, when fluorescently-labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides.

Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.

It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.

Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods. As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples of radioactive labels include 3H, 14C, 32P, and 35S.

Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.

It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays.

Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.

Amino Acid Sequences and Peptides

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide” and “protein” include glycoproteins, as well as non-glycoproteins.

Polypeptide products can be biochemically synthesized such as by employing standard solid phase techniques. Such methods include but are not limited to exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.

Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.], after which their composition can be confirmed via amino acid sequencing.

In cases where large amounts of a polypeptide are desired, it can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

The present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention, as well as polypeptides according to the amino acid sequences described herein. The present invention also encompasses homologues of these polypeptides, such homologues can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95% or more say 100% homologous to the amino acid sequences set forth below, as can be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low-complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and extension), and number of alignments shown is 50. Finally, the present invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or artificially induced, either randomly or in a targeted fashion.

It will be appreciated that peptides identified according the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified. Further details in this respect are provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated bonds (—N(CH3)-CO—), ester bonds (—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), *-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds (—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—), peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below the term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids.

Table 1 non-conventional or modified amino acids which can be used with the present invention.

Since the peptides of the present invention are preferably utilized in therapeutics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.

The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.

The peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis well known in the art, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.

Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing.

In cases where large amounts of the peptides of the present invention are desired, the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 and also as described above.

Peptide sequences which exhibit high therapeutic activity, such as by competing with wild type signaling proteins of the same signaling pathway, can be also uncovered using computational biology. Software programs useful for displaying three-dimensional structural models, such as RIBBONS (Carson, M., 1997. Methods in Enzymology 277, 25), O (Jones, T A. et al., 1991. Acta Crystallogr. A47, 110), DINO (DINO: Visualizing Structural Biology (2001); and QUANTA, INSIGHT, SYBYL, MACROMODE, ICM, MOLMOL, RASMOL and GRASP (reviewed in Kraulis, J., 1991. Appl Crystallogr. 24, 946) can be utilized to model interactions between the polypeptides of the present invention and prospective peptide sequences to thereby identify peptides which display the highest probability of binding for example to a respective ligand (e.g., IL-10). Computational modeling of protein-peptide interactions has been successfully used in rational drug design, for further details, see Lam et al., 1994. Science 263, 380; Wlodawer et al., 1993. Ann Rev Biochem. 62, 543; Appelt, 1993. Perspectives in Drug Discovery and Design 1, 23; Erickson, 1993. Perspectives in Drug Discovery and Design 1, 109, and Mauro M J. et al., 2002. J Clin Oncol. 20, 325-34.

Antibodies

“Antibody” refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad-immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab′ and F(ab)′2 fragments. The term “antibody,” as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. “Fc” portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CH1, CH2 and CH3, but does not include the heavy chain variable region.

The functional fragments of antibodies, such as Fab, F(ab′)2, and Fv that are capable of binding to macrophages, are described as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (“SCA”), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli . The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′) or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)1, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

Preferably, the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention. As used herein, the term “epitope” refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.

Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

Optionally, a unique epitope may be created in a variant due to a change in one or more post-translational modifications, including but not limited to glycosylation and/or phosphorylation, as described below. Such a change may also cause a new epitope to be created, for example through removal of glycosylation at a particular site.

An epitope according to the present invention may also optionally comprise part or all of a unique sequence portion of a variant according to the present invention in combination with at least one other portion of the variant which is not contiguous to the unique sequence portion in the linear polypeptide itself, yet which are able to form an epitope in combination. One or more unique sequence portions may optionally combine with one or more other non-contiguous portions of the variant (including a portion which may have high homology to a portion of the known protein) to form an epitope.

Display Libraries

According to still another aspect of the present invention there is provided a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 6, at least 7, at least 8, at least 9, at least 10, 10-15, 12-17, 15-20, 15-30 or 20-50 consecutive amino acids derived from the polypeptide sequences of the present invention.

Since in therapeutic applications it is highly desirable to employ the minimal and most efficacious polypeptide regions, which still exert therapeutic function, identification of such peptide regions can be effected using various approaches, including, for example, display techniques as described herein.

Methods of constructing such display libraries are well known in the art. Such methods are described in, for example, Young A C, et al., “The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes” J Mol Biol 1997 Dec. 12; 274(4):622-34; Giebel L B et al. “Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities” Biochemistry 1995 Nov. 28; 34(47):15430-5; Davies E L et al., “Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes” J Immunol Methods 1995 Oct. 12; 186(1):125-35; Jones C RT al. “Current trends in molecular recognition and bioseparation” J Chromatogr A 1995 Jul. 14; 707(1):3-22; Deng S J et al. “Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries” Proc Natl Acad Sci USA 1995 May 23; 92(11):4992-6; and Deng S J et al. “Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display” J Biol Chem 1994 Apr. 1; 269(13):9533-8, which are incorporated herein by reference.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

A “variant-treatable” disease refers to any disease that is treatable by using a splice variant of any of the therapeutic proteins according to the present invention. “Treatment” also encompasses prevention, amelioration, elimination and control of the disease and/or pathological condition. The diseases for which such variants may be useful therapeutic agents are described in greater detail below for each of the variants. The variants themselves are described by “cluster” or by gene, as these variants are splice variants of known proteins. Therefore, a “cluster-related disease” or a “protein-related disease” refers to a disease that may be treated by a particular protein, with regard to the description of such diseases below a therapeutic protein variant according to the present invention.

The term “biologically active”, as used herein, refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” refers to the capability of the natural, recombinant, or synthetic ligand, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

The term “modulate”, as used herein, refers to a change in the activity of at least one receptor mediated activity. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional or immunological properties of a ligand.

Methods of Treatment

As mentioned hereinabove the novel therapeutic protein variants of the present invention and compositions derived therefrom (i.e., peptides, oligonucleotides) can be used to treat cluster or protein-related diseases, disorders or conditions.

Thus, according to an additional aspect of the present invention there is provided a method of treating cluster or protein-related disease, disorder or condition in a subject.

The subject according to the present invention is a mammal, preferably a human which is diagnosed with one of the disease, disorder or conditions described hereinabove, or alternatively is predisposed to at least one type of the cluster or protein-related disease, disorder or conditions described hereinabove.

As used herein the term “treating” refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the above-described diseases, disorders or conditions.

Treating, according to the present invention, can be effected by specifically upregulating or alternatively downregulating the expression of at least one of the polypeptides of the present invention in the subject.

Optionally, upregulation may be effected by administering to the subject at least one of the polypeptides of the present invention (e.g., recombinant or synthetic) or an active portion thereof, as described herein (e.g., SEQ ID NO:1, 5, 9, 13, 17 or 54). However, since the bioavailability of large polypeptides may potentially be relatively small due to high degradation rate and low penetration rate, administration of polypeptides is preferably confined to small peptide fragments (e.g., about 100 amino acids). The polypeptide or peptide may optionally be administered in as part of a pharmaceutical composition, described in more detail below.

It will be appreciated that treatment of the above-described diseases according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy). Thus, treatment of malignancies using the agents of the present invention may be combined with, for example, radiation therapy, antibody therapy and/or chemotherapy.

Alternatively or additionally, an upregulating method may optionally be effected by specifically upregulating the amount (optionally expression) in the subject of at least one of the polypeptides of the present invention (e.g., SEQ ID NO: 1, 5, 9, 13, 17 or 54) or active portions thereof.

As is mentioned hereinabove and in the Examples section which follows, the biomolecular sequences of this aspect of the present invention may be used as valuable therapeutic tools in the treatment of diseases, disorders or conditions in which altered activity or expression of the wild-type gene product is known to contribute to disease, disorder or condition onset or progression. For example, in case a disease is caused by overexpression of a membrane bound-receptor, a soluble variant thereof may be used as an antagonist which competes with the receptor for binding the ligand, to thereby terminate signaling from the receptor. Examples of such diseases are listed in the Examples section which follows.

It will be appreciated that the polypeptides of the present invention may also have agonistic properties. These include increasing the stability of the ligand (e.g., IL-4), protection from proteolysis and modification of the pharmacokinetic properties of the ligand (i.e., increasing the half-life of the ligand, while decreasing the clearance thereof). As such, the biomolecular sequences of this aspect of the present invention may be used to treat conditions or diseases in which the wild-type gene product plays a favorable role, for example, increasing angiogenesis in cases of diabetes or ischemia.

Upregulating expression of the therapeutic protein or polypeptide variants of the present invention may be effected via the administration of at least one of the exogenous polynucleotide sequences of the present invention (e.g., SEQ ID NO:3, 11, 15, 19, 45), ligated into a nucleic acid expression construct (as described in greater detail hereinabove) designed for expression of coding sequences in eukaryotic cells (e.g., mammalian cells), as described above. Accordingly, the exogenous polynucleotide sequence may be a DNA or RNA sequence encoding the variants of the present invention or active portions thereof.

It will be appreciated that the nucleic acid construct can be administered to the individual employing any suitable mode of administration including in vivo gene therapy (e.g., using viral transformation as described hereinabove). Alternatively, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex-vivo gene therapy).

Such cells (i.e., which are transfected with the nucleic acid construct of the present invention) can be any suitable cells, such as kidney, bone marrow, keratinocyte, lymphocyte, adult stem cells, cord blood cells, embryonic stem cells which are derived from the individual and are transfected ex vivo with an expression vector containing the polynucleotide designed to express the polypeptide of the present invention as described hereinabove.

Administration of the ex vivo transfected cells of the present invention can be effected using any suitable route such as intravenous, intra peritoneal, intra kidney, intra gastrointestinal track, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural and rectal. According to presently preferred embodiments, the ex vivo transfected cells of the present invention are introduced to the individual using intravenous, intra kidney, intra gastrointestinal track and/or intra peritoneal administrations.

The ex vivo transfected cells of the present invention can be derived from either autologous sources such as self bone marrow cells or from allogeneic sources such as bone marrow or other cells derived from non-autologous sources. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells or tissues in immunoisolating, semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu M Z, et al., Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μm (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002; 13: 783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

It will be appreciated that the present methodology may also be effected by specifically upregulating the expression of the variants of the present invention endogenously in the subject. Agents for upregulating endogenous expression of specific splice variants of a given gene include antisense oligonucleotides, which are directed at splice sites of interest, thereby altering the splicing pattern of the gene. This approach has been successfully used for shifting the balance of expression of the two isoforms of Bcl-x [Taylor (1999) Nat. Biotechnol. 17:1097-1100; and Mercatante (2001) J. Biol. Chem. 276:16411-164171; IL-5R [Karras (2000) Mol. Pharmacol. 58:380-3871; and c-myc [Giles (1999) Antisense Acid Drug Dev. 9:213-2201.

For example, interleukin 5 and its receptor play a critical role as regulators of hematopoiesis and as mediators in some inflammatory diseases such as allergy and asthma. Two alternatively spliced isoforms are generated from the IL-5R gene, which include (i.e., long form) or exclude (i.e., short form) exon 9. The long form encodes for the intact membrane-bound receptor, while the shorter form encodes for a secreted soluble non-functional receptor. Using 2′-O-MOE-oligonucleotides specific to regions of exon 9, Karras and co-workers (supra) were able to significantly decrease the expression of the wild type receptor and increase the expression of the shorter isoforms. Design and synthesis of oligonucleotides which can be used according to the present invention are described hereinbelow and by Sazani and Kole (2003) Progress in Moleclular and Subcellular Biology 31:217-239.

Treatment can preferably effected by agents which are capable of specifically downregulating expression (or activity) of at least one of the polypeptide variants of the present invention.

Down regulating the expression of the therapeutic protein variants of the present invention may be achieved using oligonucleotide agents such as those described in greater detail below.

SiRNA molecules—Small interfering RNA (siRNA) molecules can be used to down-regulate expression of the therapeutic protein variants of the present invention.

RNA interference is a two-step process. The first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3′ overhangs [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature 409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3′ terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although the mechanism of cleavage is still to be elucidated, research indicates that each RISC contains a single siRNA and an RNase [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For more information on RNAi see the following reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat. Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25 (2002).

Synthesis of RNAi molecules suitable for use with the present invention can be effected as follows. First, the mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3′ adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA and completely abolished protein level.

Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server. Putative target sites which exhibit significant homology to other coding sequences are filtered out. Qualifying target sequences are selected as template for siRNA synthesis.

Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%. Several target sites are preferably selected along the length of the target gene for evaluation. Target sites are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.

DNAzyme molecules—Another agent capable of downregulating expression of the polypeptides of the present invention is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the polynucleotides of the present invention. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262) A general model (the “10-23” model) for the DNAzyme has been proposed. “10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].

Target sites for DNAzymes are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated.

Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 20002, Abstract 409, Ann Meeting Am Soc Gen Ther). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.

Antisense molecules—Downregulation of the polynucleotides of the present invention can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the polypeptide variants of the present invention.

The term “antisense”, as used herein, refers to any composition containing nucleotide sequences, which are complementary to a specific DNA or RNA sequence.

The term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand. Antisense molecules also include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference to the sense strand. Antisense oligonucleotides are also used for modulation of alternative splicing in vivo and for diagnostics in vivo and in vitro (Khelifi C. et al., 2002, Current Pharmaceutical Design 8:451-1466; Sazani, P., and Kole. R. Progress in Molecular and Cellular Biology, 2003, 31:217-239).

Design of antisense molecules which can be used to efficiently down-regulate expression of the polypeptides of the present invention must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.

The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett et al. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40 (1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9 (1999)].

Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al. enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gp130) in cell culture as evaluated by a kinetic PCR technique proved effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin Mol Ther 1:297-306 (1999)].

More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60 (2001)].

Thus, the current consensus is that recent developments in the field of antisense technology which, as described above, have led to the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for downregulating expression of known sequences without having to resort to undue trial and error experimentation.

Target sites for antisense molecules are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated.

Ribozymes—Another agent capable of downregulating expression of the polypeptides of the present invention is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding the polypeptide variants of the present invention. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated—WEB home page).

An additional method of regulating the expression of a specific gene in cells is via triplex forming oligonucleotides (TFOs). Recent studies have shown that TFOs can be designed which can recognize and bind to polypurine/polypirimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outlined by Maher III, L. J., et al., Science, 1989; 245:725-730; Moser, H. E., et al., Science, 1987; 238:645-630; Beal, P. A., et al, Science, 1992; 251:1360-1363; Cooney, M., et al., Science, 1988; 241:456-459; and Hogan, M. E., et al., EP Publication 375408. Modification of the oligonucleotides, such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review see Seidman and Glazer, J Clin Invest 2003; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequence correspondence:

However, it has been shown that the A-AT and G-GC triplets have the greatest triple helical stability (Reither and Jeltsch, BMC Biochem, 2002, Sep. 12, Epub). The same authors have demonstrated that TFOs designed according to the A-AT and G-GC rule do not form non-specific triplexes, indicating that the triplex formation is indeed sequence specific.

Thus for any given sequence in the gene regulatory region a triplex forming sequence may be devised. Triplex-forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression. Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFG1 and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999; 27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and the sequence- and target specific downregulation of expression of the Ets2 transcription factor, important in prostate cancer etiology (Carbone, et al, Nucl Acid Res. 2003; 31:833-43), and the pro-inflammatory ICAM-1 gene (Besch et al, J Biol Chem, 2002; 277:32473-79). In addition, Vuyisich and Beal have recently shown that sequence specific TFOs can bind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000; 28:2369-74).

Additionally, TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94). Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.

Alternatively, down regulation of the polypeptide variants of the present invention may be achieved at the polypeptide level using downregulating agents such as antibodies or antibody fragments capable of specifically binding the polypeptides of the present invention and inhibiting the activity thereof (i.e., neutralizing antibodies). Such antibodies can be directed for example, to the heterodimerizing domain on the variant, or to a putative ligand binding domain. Further description of antibodies and methods of generating same is provided below.

Alternatively, down regulation of the polypeptide variants of the present invention may be achieved using small, unique peptide sequences (e.g., of about 50-100 amino acids) which are capable of specifically binding to their target molecules (e.g., a receptor subunit) and thus prevent endogenous subunit assembly or association and therefore antagonize the receptor activity. Such peptides can be natural or synthetic peptides which are derived from the polypeptide of the present invention (e.g., SEQ ID NO:9, 77 or 164).

Pharmaceutical Compositions and Delivery Thereof

The present invention features a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic agent according to the present invention, which is preferably a therapeutic protein variant as described herein. Optionally and alternatively, the therapeutic agent could be an antibody or an oligonucleotide that specifically recognizes and binds to the therapeutic protein variant, but not to the corresponding full length known protein.

Alternatively, the pharmaceutical composition of the present invention includes a therapeutically effective amount of at least an active portion of a therapeutic protein variant polypeptide.

The pharmaceutical composition according to the present invention is preferably used for the treatment of cluster or protein-related disease, disorder or condition.

“Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.

A “disorder” is any condition that would benefit from treatment with the agent according to the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein are described with regard to specific examples given herein.

The term “therapeutically effective amount” refers to an amount of agent according to the present invention that is effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the agent may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the agent may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

The therapeutic agents of the present invention can be provided to the subject per se, or as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term “active ingredient” refers to the preparation accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).

Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Pharmaceutical compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

Immunogenic Compositions

A therapeutic agent according to the present invention may optionally be a molecule, which promotes a specific immunogenic response against at least one of the polypeptides of the present invention in the subject. The molecule can be polypeptide variants of the present invention, a fragment derived therefrom or a nucleic acid sequence encoding thereof. Although such a molecule can be provided to the subject per se, the agent is preferably administered with an immunostimulant in an immunogenic composition. An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes into which the compound is incorporated (see e.g., U.S. Pat. No. 4,235,877). Vaccine preparation is generally described in, for example, M. F. Powell and M. J. Newman, eds., “Vaccine Design (the subunit and adjuvant approach),” Plenum Press (NY, 1995).

Illustrative immunogenic compositions may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. The DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems (see below), bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the subject (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus - Calmette - Guerrin ) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked,” as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

It will be appreciated that an immunogenic composition may comprise both a polynucleotide and a polypeptide component. Such immunogenic compositions may provide for an enhanced immune response.

Any of a variety of immunostimulants may be employed in the immunogenic compositions of this invention. For example, an adjuvant may be included. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

The adjuvant composition may be designed to induce an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-.gamma, TNF.alpha., IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of an immunogenic composition as provided herein, the subject will support an immune response that includes Th1- and Th2-type responses. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffinan, Ann Rev. Immunol. 7:145-173, 1989.

Preferred adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720.

A delivery vehicle may be employed within the immunogenic composition of the present invention to facilitate production of an antigen-specific immune response that targets tumor cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmernan and Levy, Ann Rev. Med. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within an immunogenic composition (see Zitvogel et al., Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF.alpha. to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF.alpha., CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.

Dendritic cells are categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcy receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

APCs may generally be transfected with at least one polynucleotide encoding a polypeptide of the present invention, such that variant II, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to the subject, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with a polypeptide of the present invention, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule) such as described above. Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

Preferred embodiments of the present invention encompass novel naturally occurring secreted (i.e., extracellular) and non-secreted (i.e., intracellular or membranal) variants of genes and gene products, which, as is described in the Examples section which follows, play pivotal roles in disease onset and progression. As such these variants can be used for a wide range of diagnostic and/or therapeutic uses.

Diagnostic Methods

The term “marker” in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients having or predisposed to a cluster or protein-related disease, disorder or condition as compared to a comparable sample taken from subjects who do not have a such a disease, disorder or condition.

The methods for detecting these markers have many applications. For example, one marker or combination of markers can be measured to differentiate between various types of cluster or protein-related disease, disorder or condition, and thus are useful as an aid in the accurate diagnosis of cluster or protein-related disease, disorder or condition in a patient. For example, one marker or combination of markers can be measured to differentiate between various types of lung cancers, such as small cell or non-small cell lung cancer, and further between non-small cell lung cancer types, such as adenocarcinomas, squamous cell and large cell carcinomas, and thus are useful as an aid in the accurate diagnosis of lung cancer in a patient. In another example, the present methods for detecting these markers can be applied to in vitro cluster or protein-related cancers cells or in vivo animal models for cluster or protein-related cancers to assay for and identify compounds that modulate expression of these markers.

The phrase “differentially present” refers to differences in the quantity of a marker present in a sample taken from patients having cluster or protein-related disease, disorder or condition as compared to a comparable sample taken from patients who do not have such disease, disorder or condition. For example, a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays. A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present. One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided below.

As used herein the phrase “diagnostic” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay are termed “true negatives.” The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

The phrase “predisposition” used herein refers to the susceptibility to develop a disorder. A subject with a predisposition to develop a disorder is more likely to develop the disorder than a non-predisposed subject.

As used herein the phrase “diagnosing” refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The term “detecting” may also optionally encompass any of the above.

Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease.

As used herein “a biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum, milk, blood cells, tumors, neuronal tissue, organs, and also samples of in vivo cell culture constituents. It should be noted that a “biological sample obtained from the subject” may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.

As used herein, the term “level” refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention.

Typically the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual.

Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject.

Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.

Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.

A “test amount” of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a cluster or protein-related disease, disorder or condition related cancer or other UbCH10 related disease. A test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).

A “control amount” of a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a patient which does not have the cluster or protein-related disease, disorder or condition. A control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).

“Detect” refers to identifying the presence, absence or amount of the object to be detected.

A “label” includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavidin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample. The label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavidin. The label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly. For example, the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavidin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology 6:1165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.

Exemplary detectable labels, optionally and preferably for use with immunoassays, include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads. Alternatively, the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.

“Immunoassay” is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with” when referring to a protein or peptide (or other epitope), refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

In another embodiment, this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention. Preferably such antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).

In another embodiment, this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting the interaction; wherein the presence of an interaction correlates with the presence of a splice variant in the biological sample.

In another embodiment, this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.

According to another embodiment of the present invention the detection of the splice variant nucleic acid sequences in the biological sample is effected by detecting at least one nucleic acid change within a nucleic acid material derived from the biological sample; wherein the presence of the at least one nucleic acid change correlates with the presence of a splice variant nucleic acid sequence in the biological sample.

According to the present invention, the splice variants described herein are non-limiting examples of markers for diagnosing the cluster or protein-related disease, disorder or condition. Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of such a cancer, disease or pathology.

According to optional but preferred embodiments of the present invention, any marker according to the present invention may optionally be used alone or combination. Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker, for example a known marker. Furthermore, such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker. With regard to such a ratio between any marker described herein (or a combination thereof) and a known marker, more preferably the known marker comprises the “known protein” as described in greater detail below with regard to each cluster or gene.

According to other preferred embodiments of the present invention, a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof, may be featured as a biomarker for detecting the cluster or protein-related disease, disorder or condition, such that a biomarker may optionally comprise any of the above.

Non-limiting examples of methods or assays are described below.

The present invention also relates to kits based upon such diagnostic methods or assays.

NAT Assays

Detection of a nucleic acid of interest in a biological sample may also optionally be effected by NAT-based assays, which involve nucleic acid amplification technology, such as PCR, or variations thereof (e.g., real-time PCR, RT-PCR and in situ RT-PCR).

As used herein, a “primer” defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.

Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the q3 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 1989, supra).

The terminology “amplification pair” (or “primer pair”) refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions.

In one particular embodiment, amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially expressed nucleic acid. In one preferred embodiment, RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA. In another preferred embodiment, the amplification of the differentially expressed nucleic acids is carried out simultaneously. It will be realized by a person skilled in the art that such methods could be adapted for the detection of differentially expressed proteins instead of differentially expressed nucleic acid sequences.

The nucleic acid (i.e. DNA or RNA) for practicing the present invention may be obtained according to well known methods.

Oligonucleotide primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed. Optionally, the oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).

It will be appreciated that antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre-mRNA level. Essentially the ability to quantitate transcription from a splice site of interest can be effected based on splice site accessibility. Oligonucleotides may compete with splicing factors for the splice site sequences. Thus, low activity of the antisense oligonucleotide is indicative of splicing activity.

The polymerase chain reaction and other nucleic acid amplification reactions are well known in the art (various non-limiting examples of these reactions are described in greater detail below). The pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7° C., preferably less than 5° C., more preferably less than 4° C., most preferably less than 3° C., ideally between 3° C. and 0° C.

Polymerase Chain Reaction (PCR): The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al., is a method of increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification. This technology provides one approach to the problems of low target sequence concentration. PCR can be used to directly increase the concentration of the target to an easily detectable level. This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize. Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.

The length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are the to be “PCR-amplified.”

Ligase Chain Reaction (LCR or LAR): The ligase chain reaction [LCR; sometimes referred to as “Ligase Amplification Reaction” (LAR)] has developed into a well-recognized alternative method of amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes: see for example Segev, PCT Publication No. WO9001069 A1 (1990). However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.

Self-Sustained Synthetic Reaction (3SR/NASBA): The self-sustained sequence replication reaction (3SR) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection. In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5′ end of the sequence of interest. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).

Q-Beta (Qβ) Replicase: In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Qβ replicase. A previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step. However, available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C.). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.

A successful diagnostic method must be very specific. A straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Qβ systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature (i.e., >55 degrees C.). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons, PCR and LCR currently dominate the research field in detection technologies.

The basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle. The final yield of any such doubling system can be expressed as: (1+X)n=y, where “X” is the mean efficiency (percent copied in each cycle), “n” is the number of cycles, and “y” is the overall efficiency, or yield of the reaction. If every copy of a target DNA is utilized as a template in every cycle of a polymerase chain reaction, then the mean efficiency is 100%. If 20 cycles of PCR are performed, then the yield will be 220, or 1,048,576 copies of the starting material. If the reaction conditions reduce the mean efficiency to 85%, then the yield in those 20 cycles will be only 1.8520, or 220,513 copies of the starting material. In other words, a PCR running at 85% efficiency will yield only 21% as much final product, compared to a reaction running at 100% efficiency. A reaction that is reduced to 50% mean efficiency will yield less than 1% of the possible product.

In practice, routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield. At 50% mean efficiency, it would take 34 cycles to achieve the million-fold amplification theoretically possible in 20, and at lower efficiencies, the number of cycles required becomes prohibitive. In addition, any background products that amplify with a better mean efficiency than the intended target will become the dominant products.

Also, many variables can influence the mean efficiency of PCR, including target DNA length and secondary structure, primer length and design, primer and dNTP concentrations, and buffer composition, to name but a few. Contamination of the reaction with exogenous DNA (e.g., DNA spilled onto lab surfaces) or cross-contamination is also a major consideration. Reaction conditions must be carefully optimized for each different primer pair and target sequence, and the process can take days, even for an experienced investigator. The laboriousness of this process, including numerous technical considerations and other factors, presents a significant drawback to using PCR in the clinical setting. Indeed, PCR has yet to penetrate the clinical market in a significant way. The same concerns arise with LCR, as LCR must also be optimized to use different oligonucleotide sequences for each target sequence. In addition, both methods require expensive equipment, capable of precise temperature cycling.

Many applications of nucleic acid detection technologies, such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences. One method of the detection of allele-specific variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3′ end of the primer. An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence. This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.

A similar 3′-mismatch strategy is used with greater effect to prevent ligation in the LCR. Any mismatch effectively blocks the action of the thermostable ligase, but LCR still has the drawback of target-independent background ligation products initiating the amplification. Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory.

The direct detection method according to various preferred embodiments of the present invention may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.

When a sufficient amount of a nucleic acid to be detected is available, there are advantages to detecting that sequence directly, instead of making more copies of that target, (e.g., as in PCR and LCR). Most notably, a method that does not amplify the signal exponentially is more amenable to quantitative analysis. Even if the signal is enhanced by attaching multiple dyes to a single oligonucleotide, the correlation between the final signal intensity and amount of target is direct. Such a system has an additional advantage that the products of the reaction will not themselves promote further reaction, so contamination of lab surfaces by the products is not as much of a concern. Recently devised techniques have sought to eliminate the use of radioactivity and/or improve the sensitivity in automatable formats. Two examples are the “Cycling Probe Reaction” (CPR), and “Branched DNA” (bDNA).

Cycling probe reaction (CPR): The cycling probe reaction (CPR), uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.

Branched DNA: Branched DNA (bDNA), involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.

The NAT assays of the present invention also include methods of detecting at least one nucleic acid change [e.g., a single nucleotide polymorphism (SNP] in the biological sample of the present invention.

The demand for tests which allow the detection of specific nucleic acid sequences and sequence changes is growing rapidly in clinical diagnostics. As nucleic acid sequence data for genes from humans and pathogenic organisms accumulates, the demand for fast, cost-effective, and easy-to-use tests for as yet mutations within specific sequences is rapidly increasing.

A handful of methods have been devised to scan nucleic acid segments for mutations or nucleic acid changes. One option is to determine the entire gene sequence of each test sample (e.g., a bacterial isolate). For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g., PCR reaction products). This avoids the time and expense associated with cloning the segment of interest. However, specialized equipment and highly trained personnel are required, and the method is too labor-intense and expensive to be practical and effective in the clinical setting.

In view of the difficulties associated with sequencing, a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel. A more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.

Restriction fragment length polymorphism (RFLP): For detection of single-base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing. For example, if a mutation of interest happens to fall within a restriction recognition sequence, a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).

Single point mutations have been also detected by the creation or destruction of RFLPs. Mutations are detected and localized by the presence and size of the RNA fragments generated by cleavage at the mismatches. Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the “Mismatch Chemical Cleavage” (MCC). However, this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory.

RFLP analysis suffers from low sensitivity and requires a large amount of sample. When RFLP analysis is used for the detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease. Moreover, the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations. Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites.

A handful of rare-cutting restriction enzymes with 8 base-pair specificities have been isolated and these are widely used in genetic mapping, but these enzymes are few in number, are limited to the recognition of G+C-rich sequences, and cleave at sites that tend to be highly clustered. Recently, endonucleases encoded by group I introns have been discovered that might have greater than 12 base-pair specificity, but again, these are few in number.

Allele specific oligonucleotide (ASO): If the change is not in a recognition sequence, then allele-specific oligonucleotides (ASOs), can be designed to hybridize in proximity to the mutated nucleotide, such that a primer extension or ligation event can bused as the indicator of a match or a mis-match. Hybridization with radioactively labeled allelic specific oligonucleotides (ASO) also has been applied to the detection of specific point mutations. The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles. The ASO approach applied to PCR products also has been extensively utilized by various researchers to detect and characterize point mutations in ras genes and gsp/gip oncogenes. Because of the presence of various nucleotide changes in multiple positions, the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations.

With either of the techniques described above (i.e., RFLP and ASO), the precise location of the suspected mutation must be known in advance of the test. That is to say, they are inapplicable when one needs to detect the presence of a mutation within a gene or sequence of interest.

Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE): Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes. One of these methods, termed “Denaturing Gradient Gel Electrophoresis” (DGGE) is based on the observation that slightly different sequences will display different patterns of local melting when electrophoretically resolved on a gradient gel. In this manner, variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of mutations in the target sequences because of the corresponding changes in their electrophoretic mobilities. The fragments to be analyzed, usually PCR products, are “clamped” at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands. The attachment of a GC “clamp” to the DNA fragments increases the fraction of mutations that can be recognized by DGGE. Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature. Modifications of the technique have been developed, using temperature gradients, and the method can be also applied to RNA:RNA duplexes.

Limitations on the utility of DGGE include the requirement that the denaturing conditions must be optimized for each type of DNA to be tested. Furthermore, the method requires specialized equipment to prepare the gels and maintain the needed high temperatures during electrophoresis. The expense associated with the synthesis of the clamping tail on one oligonucleotide for each sequence to be tested is also a major consideration. In addition, long running times are required for DGGE. The long running time of DGGE was shortened in a modification of DGGE called constant denaturant gel electrophoresis (CDGE). CDGE requires that gels be performed under different denaturant conditions in order to reach high efficiency for the detection of mutations.

A technique analogous to DGGE, termed temperature gradient gel electrophoresis (TGGE), uses a thermal gradient rather than a chemical denaturant gradient. TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field. TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel.

Single-Strand Conformation Polymorphism (SSCP): Another common method, called “Single-Strand Conformation Polymorphism” (SSCP) was developed by Hayashi, Sekya and colleagues and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations.

The SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run. This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.

Dideoxy fingerprinting (ddF): The dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations. The ddF technique combines components of Sanger dideoxy sequencing with SSCP. A dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).

In addition to the above limitations, all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed. For the direct sequencing approach, sequences of greater than 600 base pairs require cloning, with the consequent delays and expense of either deletion sub-cloning or primer walking, in order to cover the entire fragment. SSCP and DGGE have even more severe size limitations. Because of reduced sensitivity to sequence changes, these methods are not considered suitable for larger fragments. Although SSCP is reportedly able to detect 90% of single-base substitutions within a 200 base-pair fragment, the detection drops to less than 50% for 400 base pair fragments. Similarly, the sensitivity of DGGE decreases as the length of the fragment reaches 500 base-pairs. The ddF technique, as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA that can be screened.

Reverse dot blot: This technique uses labeled sequence specific oligonucleotide probes and unlabeled nucleic acid samples. Activated primary amine-conjugated oligonucleotides are covalently attached to carboxylated nylon membranes. After hybridization and washing, the labeled probe, or a labeled fragment of the probe, can be released using oligomer restriction, i.e., the digestion of the duplex hybrid with a restriction enzyme. Circular spots or lines are visualized colorimetrically after hybridization through the use of streptavidin horseradish peroxidase incubation followed by development using tetramethylbenzidine and hydrogen peroxide, or via chemiluminescence after incubation with avidin alkaline phosphatase conjugate and a luminous substrate susceptible to enzyme activation, such as CSPD, followed by exposure to x-ray film.

It will be appreciated that advances in the field of SNP detection have provided additional accurate, easy, and inexpensive large-scale SNP genotyping techniques, such as Pyrosequencing™, Acycloprime™, dynamic allele-specific hybridization (DASH, Howell, W. M. et al., 1999. Dynamic allele-specific hybridization (DASH). Nat. Biotechnol. 17: 87-8), microplate array diagonal gel electrophoresis [MADGE, Day, I. N. et al., 1995. High-throughput genotyping using horizontal polyacrylamide gels with wells arranged for microplate array diagonal gel electrophoresis (MADGE). Biotechniques. 19: 830-5], the TaqMan system (Holland, P. M. et al., 1991. Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA. 88: 7276-80), as well as various DNA “chip” technologies such as the GeneChip microarrays (e.g., Affymetrix SNP chips) which are disclosed in U.S. Pat. No. 6,300,063 to Lipshutz, et al. 2001, which is fully incorporated herein by reference, Genetic Bit Analysis (GBA™) which is described by Goelet, P. et al. (PCT Appl. No. 92/15712), peptide nucleic acid (PNA, Ren B, et al., 2004. Nucleic Acids Res. 32: e42) and locked nucleic acids (LNA, Latorra D, et al., 2003. Hum. Mutat. 22: 79-85) probes, Molecular Beacons (Abravaya K, et al., 2003. Clin Chem Lab Med. 41: 468-74), intercalating dye [Germer, S. and Higuchi, R. Single-tube genotyping without oligonucleotide probes. Genome Res. 9:72-78 (1999)], FRET primers (Solinas A et al., 2001. Nucleic Acids Res. 29: E96), AlphaScreen (Beaudet L, et al., Genome Res. 2001, 11(4): 600-8), SNPstream (Bell P A, et al., 2002. Biotechniques. Suppl.: 70-2, 74, 76-7), Multiplex minisequencing (Curcio M, et al., 2002. Electrophoresis. 23: 1467-72), SnaPshot (Turner D, et al., 2002. Hum Immunol. 63: 508-13), MassEXTEND (Cashman J R, et al., 2001. Drug Metab Dispos. 29: 1629-37), GOOD assay (Sauer S, and Gut I G. 2003. Rapid Commun. Mass. Spectrom. 17: 1265-72), Microarray minisequencing (Liljedahl U, et al., 2003. Pharmacogenetics. 13: 7-17), arrayed primer extension (APEX) (Tonisson N, et al., 2000. Clin. Chem. Lab. Med. 38: 165-70), Microarray primer extension (O'Meara D, et al., 2002. Nucleic Acids Res. 30: e75), Tag arrays (Fan J B, et al., 2000. Genome Res. 10: 853-60), Template-directed incorporation (TDI) (Akula N, et al., 2002. Biotechniques. 32: 1072-8), fluorescence polarization (Hsu T M, et al., 2001. Biotechniques. 31: 560, 562, 564-8), Colorimetric oligonucleotide ligation assay (OLA, Nickerson D A, et al., 1990. Proc. Natl. Acad. Sci. USA. 87: 8923-7), Sequence-coded OLA (Gasparini P, et al., 1999. J. Med. Screen. 6: 67-9), Microarray ligation, Ligase chain reaction, Padlock probes, Rolling circle amplification, Invader assay (reviewed in Shi M M. 2001. Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem. 47: 164-72), coded microspheres (Rao K V et al., 2003. Nucleic Acids Res. 31: e66) and MassArray (Leushner J, Chiu N H, 2000. Mol Diagn. 5: 341-80).

According to a presently preferred embodiment of the present invention the step of searching for any of the nucleic acid sequences described here, in tumor cells or in cells derived from a cancer patient is effected by any suitable technique, including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self-sustained synthetic reaction, QP-Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis, dideoxy fingerprinting, Pyrosequencing™, Acycloprime™, and reverse dot blot.

Detection may also optionally be performed with a chip or other such device. The nucleic acid sample which includes the candidate region to be analyzed is preferably isolated, amplified and labeled with a reporter group. This reporter group can be a fluorescent group such as phycoerythrin. The labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station. For example, Manz et al. (1993) Adv in Chromatogr 1993; 33:1-66 describe the fabrication of fluidics devices and particularly microcapillary devices, in silicon and glass substrates.

Once the reaction is completed, the chip is inserted into a scanner and patterns of hybridization are detected. The hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.

Preferably, the detection of at least one nucleic acid change and/or the splice variant sequence of the present invention is effected in a biological sample containing RNA molecules using, for example, RT-PCR or in situ RT-PCR.

RT-PCR analysis: This method uses PCR amplification of relatively rare RNAs molecules. First, RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers. Then by applying gene specific primers and Taq DNA polymerase, a PCR amplification reaction is carried out in a PCR machine. Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semi-quantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification product to known controls.

In situ RT-PCR stain: This method is described in Nuovo G J, et al. [Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P, et al. [Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-251. Briefly, the RT-PCR reaction is performed on fixed cells by incorporating labeled nucleotides to the PCR reaction. The reaction is carried on using a specific in situ RT-PCR apparatus such as the laser-capture microdissection PixCell I LCM system available from Arcturus Engineering (Mountainview, Calif.).

It will be appreciated that when utilized along with automated equipment, the above described detection methods can be used to screen multiple samples for a disease and/or pathological condition both rapidly and easily.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

The first line of defense against infectious agents is comprised primarily of polymorphonuclear granulocytes, macrophages, natural killer cells and cytotoxic lymphocytes. GCSF, a central mediator of the endogenous response to infection and inflammation, plays a critical role in the process of hematopoiesis, regulating the proliferation, differentiation and survival of neutrophils and neutrophilic progenitor cells. It is produced mainly by hematopoietic cells, such as monocytes/macrophages and lymphocytes. Other cells, such as fibroblast, endothelial cells, astrocytes and bone marrow stromal cells, can also produce GCSF following activation by LPS, IL-1 or TNF-α. Indeed, GCSF production is sharply increased in response to bacterial infection and cell-mediated immune responses, supporting its role in vivo as a host defense against microorganisms. In vitro, GCSF exhibits stimulation of neutrophil production from precursor cells and enhancement of mature neutrophil function as augmentation of their antibody-dependant cellular cytotoxicity (ADCC). The dual action of GCSF in vitro, suggested that it would be useful clinically to stimulate hematopoietic recovery in situations of reduced bone marrow capacity or to enhance the ability to resolve infections in immunocompromised hosts. In its native form, the GCSF protein is O-glycosylated with a molecular mass of approximately 20 kD. It is a member of a family of cytokines that have a four-α-helical bundle structure which contribute importantly to its three-dimensional structure. GCSF mediates its biological actions by binding to a specific cell surface receptor, the GCSF-R, which is expressed on neutrophils, their precursors and some leukemic cell lines. Binding of GCSF causes receptor dimerization and activation of signaling cascades such as the Jak-STAT and mitogen-activated kinase pathways. The receptor has no intrinsic tyrosine kinases activity but rather it activates a number of cytoplasmic tyrosine kinases that initiate the cascade of signaling events. There are four tyrosine residues in the cytoplasmic region of the GCSF-R that are rapidly phosphorylated following ligand binding and have been shown to have specific roles in mediating the various activities of GCSF (Basu et al. 2002. International Journal of molecular Medicine. 10:3-10; Layton J. E. 1992. Growth Factors Vol. 6, Pp. 17-186; Young et al. 1997. Protein Science. 6:1228-1236; Layton et al. 1999. The Journal of Biological Chemistry. Vol. 274, No. 25, Pp. 17445-17451; Bishop et al. 2001. The Journal of Biological Chemistry. Vol. 276, No. 36, Pp. 33465-33470; Hubei et al. 2003. Ann Hematol. 82:207-213; Kuga et al. 1989. Biochemical and biophysical research communications. Vol. 159, No. 1. Pp 103-111; Clark-Lewis et al. 1988. The Journal of Immunology. Vol. 141, No. 3, Pp 881-889).

Clinical Application

Neutropenia (low neutrophils in the blood) is still the leading factor limiting the use of chemotherapy for the treatment of neoplastic diseases and a major cause of morbidity and mortality following hematopoietic stem cell transplantation. GCSF is widely employed clinically to treat cancer patients undergoing chemotherapy in order to alleviate the depression of white blood cells levels produced by cytotoxic therapeutic agents. It has been also used to accelerate hematopoietic recovery after transplantation and therefore reduce the risks of serious infection. Use of this cytokine reduces the duration of neutropenia, enhances hematopoietic reconstitution and increases the yield of the progenitor cells. Since GCSF treatment leads to rapid expansion of bone marrow cellularity and the appearance of progenitors in peripheral blood, it has been used to mobilize CD34 + hematopoietic stem cells from the marrow to the blood (peripheral blood stem cells) for use in hematopoietic transplantation. Approved pharmaceutical forms of GCSF for human use include a recombinant nonglycosylated protein expressed in Escherichia coli (filgrastim, produced by Amgen, Thousand Oaks, Calif., USA) and a glycosylated form expressed in Chinese hamster ovary cells (lenograstim, produced by Chugai Pharmaceuticals, Tokyo, Japan). Both forms have similar biological activities and bioavailability following subcutaneous or intravenous administration.

GCSF Splice Variants Structure

A brief description is now provided of GCSF splice variants according to the present invention. GCSF splice variant T3 [HUMGCSF_P4 (SEQ ID NO:1); HUMGCSF_T3 (SEQ ID NO:3), FIGS. 1 a and b , respectively, shown according to the name of the cluster, HUMGCSF) results from alternative splicing of the GCSF gene, thus causing a retention of intron 2 (according to refsec GenBank Accession No. NM — 172220), leading to an insertion of a stop codon and the generation of a truncated protein ( FIGS. 2-4 ; FIG. 2 shows the EST support for the variant T3; FIG. 3 shows the alignment of the variant T3 (with the name HUMGCSF_P4) and the known protein; FIG. 4 shows the known protein [e.g., wild type (WT); CSF3_HUMAN—SEQ ID NO:1281 structure as compared to variant T3 according to the present invention). GCSF splice variant T3 encodes a 104 amino acids long protein, which contains amino acids 14-101 of WT GCSF and a unique sequences of six amino acids at the C-terminus of the protein (VSVRKG—SEQ ID NO:2). This splice variant has a novel signal P (signal peptide) and contains part of the IL6/GCSF/MGF family domain (residues 47-97, out of 51-202 of the WT or known protein sequence).

Comparison Report Between HUMGCSF_P4 and CSF3_HUMAN

1. An isolated chimeric polypeptide HUMGCSF_P4 (SEQ ID NO:1), comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MSPEPALSP corresponding to amino acids 1-9 of HUMGCSF_P4 (SEQ ID NO:1), a second amino acid sequence being at least 90% homologous to ALQLLLWHSALWTVQEATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKL corresponding to amino acids 14-65 of CSF3_HUMAN (SEQ ID NO:128), which also corresponds to amino acids 10-61 of HUMGCSF_P4 (SEQ ID NO:1), a third amino acid sequence being at least 90% homologous to CATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQL corresponding to amino acids 69-104 of CSF3_HUMAN (SEQ ID NO:128), which also corresponds to amino acids 62-97 of HUMGCSF_P4 (SEQ ID NO:1), and a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VSVRKG (SEQ ID NO:2) corresponding to amino acids 98-103 of HUMGCSF_P4 (SEQ ID NO:1), wherein said first amino acid sequence, second amino acid sequence, third amino acid sequence and fourth amino acid sequence are contiguous and in a sequential order.

2. An isolated head of HUMGCSF_P4 (SEQ ID NO:1), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MSPEPALSP of HUMGCSF_P4 (SEQ ID NO:1).

3. An isolated chimeric polypeptide for an edge portion of HUMGCSF_P4 (SEQ ID NO:1), comprising a polypeptide having a length “n”, wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise LC, having a structure as follows: a sequence starting from any of amino acid numbers 61-x to 61; and ending at any of amino acid numbers 62+((n−2)−x), in which x varies from 0 to n−2.

4. An isolated polypeptide for a tail of HUMGCSF_P4 (SEQ ID NO:1), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSVRKG (SEQ ID NO:2) in HUMGCSF_P4 (SEQ ID NO:1).

Therapeutic Potential of GCSF Splice Variant

GCSF is widely employed clinically to treat cancer patients undergoing chemotherapy in order to alleviate the depression of white blood cells levels and to accelerate hematopoietic recovery after transplantation. Furthermore, much interest has focused on the use of GCSF to mobilize CD34 + hematopoietic stem cells from the marrow to the blood for use in hematopoietic transplantation. In addition, human recombinant GCSF (HR-GCSF) can be used to stimulate a sustained elevation of circulating neutrophils in lactating dairy cows which suffer from mastitis (Cullor J S, et al., 1990; Vet. Clin. Pathol. 19: 9-12).

GCSF is currently administered as frequent injections of significant quantities of the cytokine throughout the course of the treatment. In addition, GCSF requires stringent formulation and storage conditions. Much effort was placed in developing alternative or improved molecules that demonstrate cytokine function but have superior pharmacological properties. GCSF splice variants according to the present invention may fulfill these requirements, exhibiting an increased stability while retaining part or all of the biological activity of GCSF.

Thus, the present inventors uncovered a therapeutic agent which can be used to: (i) increase white blood cell counts (e.g., neutrophils, neutrophil progenitor cells) in a subject in need thereof [e.g., a subject undergoing chemotherapy, hematopoietic stem cell transplantation, a subject suffering from mastitis (such as a lactating cow)], and (ii) mobilize CD24+ hematopietic stem cells from the bone marrow to the peripheral blood. Such an agent is a polypeptide homologous to the GCSF variant of the present invention (SEQ ID NO:1), and/or a polynucleotide homologous to SEQ ID NO:3.

It will be appreciated that such an agent can be administered or provided to an individual in need thereof per se or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

Tumor Necrosis Factor Receptor-3 (TNR3)/Lymphotoxin-β Receptor (LT-βR)

Lymphotoxin-β receptor (LT-βR) is a member of the tumor necrosis factor receptor (TNFR) superfamily and is expressed on the surface of most of cell types, including cells of epithelial and myeloid lineages but not on T and B lymphocytes. LT-βR can specifically bind two ligands: the membrane form of lymphotoxin, LT-α1/β2, which is uniquely expressed on activated lymphoid cells and LIGHT, a member of the TNF superfamily, which is induced on the cell surface during T cell activation. LT-βR has been speculated to play an essential role in the development of lymphoid organs. Thus, LT-β knock-out mice exhibit impaired lymph node development and loss of splenic architecture. In addition, LT-βR deficient mice were found to lack Peyer's patches, colon-associated lymphoid tissues and all lymph nodes. Moreover, stimulation of LT-βR on certain cell lines by LT-α1/β2 or anti-LTβR antibodies was found to induce cell death, chemokine secretion, and activation of nuclear factor κB (NFκB), suggesting an important biological function for LT-βR in mature individuals.

Like other members of the TNF receptor family, the cytoplasmic domain (CD) of LT-βR does not include consensus sequences characteristic of enzymatic activity. Thus, signaling is thought to be mediated by the proteins interacting with LT-βR such as the two serine/threonine protein kinases, p50 and p80 and the two members of the TNF receptor-associated factor (TRAF) family, TRAF3 and TRAF5, which specifically associate with the LT-βR(CD). Further study has indicated that TRAF3 plays an important role in mediating LT-βR-induced apoptosis, whereas TRAF5 involves in the activation of NFκB. On the other hand, several members of the TNFR superfamily (such as TNFRI, Fas, DR3, DR4, and DR5) contain a common motif, the death domain, in their cytoplasmic region that initiate the activation of caspase cascades to execute apoptosis. LT-βR(CD) does not contain a death domain, but signaling through LT-βR can also induce apoptosis. It was shown that the cytoplasmic domain of TNFRI can self-associate through its death domain, therefore overexpression of TNFRI or of the cytoplasmic domain thereof can induce receptor clustering, a crucial step for subsequent intracellular signaling. Despite the absence of the death domain, the LT-βR(CD) is capable of self-association. Thus, overexpression of LT-βR is sufficient to trigger apoptosis without the need for ligand conjugation (Tamada et al. 2002. The Journal of Clinical Investigation. Vol. 109, No. 4, Pp. 549-557; Shao et al. 2003. Eur. J. Immunol. 33:1736-1743; Ettinger et al. 1996. Proc. Natl. Acad. Sci. USA. Vol. 93, Pp. 13102-13107; Wu et al. 1999. The Journal of Biological Chemistry. Vol. 274, No. 17, Pp. 11868-11873; Hehlgans et al. 2002. Cancer Research 62:4034-4040).

Clinical Application

It has been shown that signaling through LT-βR induced cell death in some human adenocarcinoma tumor lines (HT-29 and WiDr) in the presence of IFN-γ. Combined in vivo treatment of human adenocarcinoma cells (WiDr), which form solid tumors in immunocompromised mice, with an agonistic anti-LT-βR antibody and human IFN-γ resulted in tumor growth arrest. Contrary to these findings, it has been shown that activation of LT-βR on fibrosarcoma tumor cells is necessary for angiogenesis and solid tumor growth. Prevention of LT-α1/β2-LT-βR signaling, by the release of LTβR-Fc from the tumor cells, inhibited tumor angiogenesis and neovascularization, and resulted in tumor growth arrest in mice. In addition, LT-βR activation on the tumor cells induced enhanced release of MIP-2, an angiogenic CXC chemokine. Thus, the interaction of activated LT-α1/β2-carrying lymphocytes with LT-βR-expressing tumor cells can initiate a novel pro-angiogenic pathway, leading to organized tumor tissue development. In addition to its modulation of tumor growth, LT-βR was shown to be involved in immune regulation. In vivo blockade of LIGHT and LTα1/β2 by administration of soluble lymphotoxin β receptor-Ig (LTβR-Ig) inhibited the cytotoxic T lymphocyte (CTL) response to host antigenic disparities and ameliorated lethal graft-versus-host disease (GVHD) in a B6 to BDF1 mouse model. In addition, it has been shown that treatment of rodents with the fusion protein, LT-βR-Ig, prevents the development of autoimmune diseases as insulitis and uveitis.

TNR3-LT-βR Splice Variant Structure

A brief description is now provided of TNR3-LT-βR splice variants according to the present invention. TNR3 splice variant transcript — 19 (SEQ ID NOs:5 and 7, FIGS. 5 a - b ; FIG. 5 a shows the nucleic acid sequence with the name of the cluster, HUMTNFRRP, with start and stop codons marked in bold and underlined; FIG. 5 b shows the corresponding amino acid sequence encoded by this nucleic acid sequence, with the protein name HUMTNFRRP_P14 and with the unique region highlighted) results from alternative splicing of the TNR3 gene, thus causing the extension of exon 4, leading to an insertion of a stop codon and the generation of a truncated protein [ FIGS. 6-8 ; FIG. 6 shows EST support for the variant; FIG. 7 shows the alignment of the variant protein HUMTNFRRP_P14 with a portion of the known protein, TNR3_HUMAN; FIG. 8 compares the structure of the WT or known TNFR3 protein to the variant protein (shown with the name T19)]. This TNR3 splice variant encodes a 166 amino acids long protein which contains the N-terminal signal sequence (residues 1-30), three TNFR CYS repeats (out of four of the WT or known protein) and a unique sequence of 9 amino acids at the C-terminus of the protein (SEQ ID NO:6). It is predicated to be a secreted protein due to the fact that it lacks the transmembrane domain of the WT or known protein.

Comparison Report Between HUMTNFRRP_P14 (SEQ ID NO:5) and TNR3_HUMAN (SEQ ID NO:129)

1. An isolated chimeric polypeptide HUMTNFRRP_P14 (SEQ ID NO:5), comprising a first amino acid sequence being at least 90% homologous to MLLPWATSAPGLAWGPLVLGLFGLLAASQPQAVPPYASENQTCRDQEKEYY EPQHRICCSRCPPGTYVSAKCSRIRDTVCATCAENSYNEHWNYLTICQLCRPC DPVMGLEEIAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTEAEL K corresponding to amino acids 1-157 of TNR3_HUMAN (SEQ ID NO:129), which also corresponds to amino acids 1-157 of HUMTNFRRP_P14 (SEQ ID NO:5), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GQRSLRGWM (SEQ ID NO:6) corresponding to amino acids 158-166 of HUMTNFRRP_P14 (SEQ ID NO:5), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMTNFRRP_P14 (SEQ ID NO:5), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GQRSLRGWM (SEQ ID NO:6) in HUMTNFRRP_P14 (SEQ ID NO:5).

The Therapeutic Potential of TNR3-LT-βR Splice Variant

TNR3 splice variant T19 encodes a soluble receptor which contains three TNFR CYS repeats (out of four of the WT or known protein). It can inhibit TNR3 signaling by competing on the ligand with the membrane-bound receptor, thus preventing LT-α1/β2 from binding to the cell surface receptor and activating it. A soluble form of TNR3 was shown already to bind LT-α1/β2 in vitro. Blocking LTαβ/TNR3 interactions was shown in vivo by administration of TNR3-Fc fusion protein or by the creation of mice which constitutively express a soluble murine TNR3-human IgG1 (Fc) transgene. Blocking TNR3 signaling could have important therapeutic potential for the treatment of cancer, graft-vs-host disease and autoimmune diseases, such as rheumatoid arthritis, Crohn's disease, insulitis and uveitis.

Thus, the present inventors uncovered a therapeutic agent which can be used to: (i) inhibit or prevent the binding of LT-α1/β2 to its TNR3 receptor in vivo or ex vivo, (ii) prevent tumor growth (e.g., solid tumor, such as fibrisarcoma) by preventing the activation of LT-βR, (iii) prevent and/or treat graft-versus-host disease (GVHD) by inhibition of the cytotoxic T lymphocyte (CTL) response, (iv) prevent and/or treat autoimmune diseases (e.g., insulitis, uveitis). Such an agent is a polypeptide homologous to the TNR3 splice variant T19 of the present invention (SEQ ID NO:5) and/or a polynucleotide homologous to SEQ ID NO:7.

It will be appreciated that such an agent can be administered or provided to an individual in need thereof per se or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

Interleukin-4 Receptor (IL-4R)

IL-4 is a pleiotropic and multifunctional cytokine produced by activated T cells, mast cells and basophils. IL-4 plays a critical role in regulating the outcome of an immune response by facilitating Th2 cell differentiation and suppressing the differentiation of IFN-γ-producing CD4 + T cells, thereby favoring humoral immune responses. The other important function of IL-4 is the regulation of immunoglobulin class-switching. It induces class-switching to IgE and IgG4 in human B cells, suggesting a preeminent role of IL-4 in the regulation of allergic conditions. IL-4 also exerts a wide variety of other effects on hematopoietic and nonhematopoietic cells. It enhances the expression of CD23 and class II MHC molecules in B cells and upregulates surface expression of the receptor complex for IL-4. On vascular endothelial cells, IL-4 together with TNF induces the expression of VCAM-1 (vascular cell adhesion molecule 1) and downregulates the expression of E-selectin, thereby changing the adhesive characteristics of endothelial cells and facilitating tissue infiltration by allergic inflammatory cells, such as eosinophils.

IL-4 receptors (IL-4R) are expressed on hematopoietic cells and a range of nonhematopoietic cells including epithelial, endothelial, muscle, fibroblast and liver cells. On hematopoietic cells, the receptor complex for IL-4 is composed of a 140 kDa high-affinity ligand-binding chain, the IL-4-receptor a chain (IL-4Rα) and the so-called common γ chain (γC) that is shared between IL-2, IL-7, IL-9 and IL-15. In contrast, in non-hematopoietic cells, the predominant accessory chain of the IL-4 receptor complex is IL-13Rα1. Furthermore, the receptor complex for IL-13 consists of various combinations of the IL-4Rα, IL-13Rα1 and IL-13Rα2. This may explain the redundancy in biological responses mediated by IL-4 and IL-13. Both IL-4 and IL-13 have been implicated in allergic diseases, probably through redundant and independent pathways. Although homodimerized IL-4Rα can generate biological signals within the cell, physiologic signaling requires heterodimerization of IL-4Rα and the accessory chain (γC). Neither IL-4Rα nor γC contains intrinsic kinase activities; rather the IL-4R requires receptor-associated kinases for the initiation of signal transduction.

Three members of the Janus kinase (Jak) family-Jak-1, Jak-2 and Jak-3 have been shown to be activated in response to IL-4R engagement and to associate with the components of the receptor complex for IL-4. Jak-1 has been proposed to bind IL-4Rα whereas Jak-3 associates with the γC chain. IL-4-IL-4R engagement results in tyrosine phosphorylation of Jak-1 and Jak-3, leading to tyrosine phosphorylation of IL-4Rα itself, a process occurring immediately after IL-4R engagement. Five conserved tyrosine residues (Tyr497, Tyr575, Tyr603, Tyr631 and Tyr713) that can be potentially phosphorylated are present in the cytoplasmic domain of IL-4Rα. Following tyrosine phosphorylation, these conserved tyrosine residues become potential docking sites for downstream signaling molecules containing Src-homology-domain 2 (SH2) or phosphotyrosine-binding domains (Mueller et al. 2002. Biochimica et Biophysica Acta. 1592:237-250; Nelms et al. 1999. Annu. Rev. Immunol. 17:701-738; Pan et al. 1999. Current Opinion in Immunology. 11:615-620; Gessner et al. 1999/2000. Immunobiology. 201, 285-307).

IL-4R Splice Variants-Structure

The present inventors uncovered a novel IL-4R isoform by applying the LEADS clustering and assembly algorithm and the annotation process, as described above.

IL-4R splice variant T5 (amino acid sequence—SEQ ID NO:9, nucleic acid sequence—SEQ ID NO:11, FIGS. 9 a - b ) results from an alternative splicing of the IL-4R gene, thus introducing a new exon, named 4a′, between exons 4 and 5, leading to an insertion of a stop codon and the generation of a truncated protein (FIGS. 10 -11). IL-4R splice variant T5 encodes a 126 amino acids long protein which contains the N-terminal signal sequence (residues 1-25), the complete CRIA domain and a unique sequence of 5 amino acids at the C-terminus of the protein (SEQ ID NO:10). Since the new IL-4R variant lacks the transmembrane domain, it is predicated to be a secreted protein.

Therapeutic Applications for the IL-4R Splice Variants of the Present Invention

Since IL-4Rα is an independent high affinity IL-4 binding subunit, the new secreted form of IL-4Rα (splice variant T5; SEQ ID NO:9), can serve as a powerful antagonist of the IL-4/IL-4R interaction since it contains the complete CRIA domain of IL-4Rα it can compete with the membrane-bound receptor on the ligand and prevent the activation of the membrane-bound IL-4R receptor. Indeed, in a murine model of allo-transplantation, the recombinant extracellular domain of IL-4R was found to block IL-4 functions both in vitro and in vivo.

IL-4-IL4R signaling pathways play a major role in the pathogenesis of allergic diseases. Moreover, naturally occurring mutations of the IL-4Rα chain have been identified and implicated in a genetic predisposition for atopic asthma. Blocking of IL-4 signaling could therefore have an important therapeutic potential for the treatment of asthma and other allergic disorders. In addition to its role in allergic disorders, IL-4R signaling was shown to be involved in autoimmune diseases and in organ transplantation. Recently, it has been shown that IL-4 may serve multiples roles in the development of lupus. Evidence for a novel role for IL-4 in the development of lupus nephritis comes from recent studies, which suggest that IL-4 may directly promote extracellular matrix deposition in the glomeruli. Blockage of IL-4 signaling may ameliorates glomerulosclerosis and prevents the development of end-stage renal disease and in general might have a therapeutic potential in the treatment of lupus, organ transplant rejection and graft-vs-host diseases.

Thus, the present inventors uncovered a therapeutic agent which can be used to: (i) prevent the association between IL4 and IL4R, (ii) treat a disorder associated with IL-4-IL-4R signaling such as asthma (e.g., atopic asthma), allergic disorder, autoimmune diseases (e.g., lupus nephritis), organ transplantation rejection, graft-vs-host disease by preventing the association between IL-4 and IL-4R. Such an agent is a polypeptide homologous to the IL-4Rα splice variant of the present invention (SEQ ID NO:9), and/or a polynucleotide homologous to SEQ ID NO:11.

It will be appreciated that such an agent can be administered or provided to an individual in need thereof per se or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

Integrin Alpha-V

The integrin family is composed of 15 α and 8 β subunits that form over twenty different αβ heterodimeric combinations on cell surfaces. Integrins recognize extracellular matrix (ECM) proteins and cell surface immunoglobulin family molecules through short peptide sequences. Several integrins (e.g., αvβ3, α5β1, αIIbβ3) strongly interact with the tripeptide Arg-Gly-Asp (RGD) sequence within the context of specific ECM or cell surface proteins. While some integrins recognize only a single ECM protein ligand (e.g. α5β1 recognizes only fibronectin), others can bind to several ligands (e.g., αvβ3 binds vitronectin, fibronectin, osteopontin, fibrinogen, denatured or proteolysed collagen and other matrix proteins). The integrin-mediated adhesion of cells to the ECM leads to bi-directional intracellular signaling events that can regulate cell survival, proliferation and migration. In contrast, inhibition of integrin-ligands interactions suppresses cellular growth or induces apoptotic cell death.

Integrin αvβ3, the most promiscuous member of the integrin family, exhibits no expression on normal tissues such as epithelial cells and very low levels on resting vascular, uterine smooth muscle, endothelium, certain activated leukocytes, macrophages and osteoclasts. On the other hand, it is expressed on tumor cells including late-stage glioblastomas, ovarian carcinoma and melanomas. Thus, integrin αvβ3 was found to contribute to the progression of melanoma by regulating melanoma cell proliferation, survival and metastases. On endothelium cells, integrin αvβ3 involves in the angiogenic process in several ways. It regulates cell adhesion to the matrix, transmit signals to the cell nucleus and is exhibits a pro-angiogenic effect by co-operating with VEGFR-2 receptor through the activation of cell signaling and the regulation of cell cycle gene expression.

Several integrin β subunits are involved in angiogenesis processes. For example, the β3 and β5 chains which associate with the αv chain. These subunits share 53% homology, however their ligand specificities are different. While αvβ3 prefers the binding to osteopontin, the αvβ5 prefers the binding to vitronectin. In fact, two different cytokine-dependent pathways participate in the activation of these integrins. While the αvβ3 pathway involves basic FGF (FGF-2) or TNF-α, αvβ5 uses VEGF, TGF-α, or PMA. Since the integrin αv subunit is widely expressed on most cell types and associates with several different β subunits, the expression of αvβ3 is likely to be regulated by the transcription of the β subunit. Other angiogenesis related αv integrin complexes include the αvβ1 which is associated with brain blood vessels and cell migration in squamous cell carcinoma; αvβ8, identified on tumor cells; and αvβ6 which induces secretion of MMP-2 in colon cancer and is important in colon cancer progression (Kerr et al. 2000. Exp. Opin. Invest. Drugs 9(6): 1271-1279; Tucker G. C. 2003. Current Opinion in Investigational Drugs. 4(6): 722-731; Mould et al. 2000. The Journal of Biological Chemistry. Vol. 275, No. 27, Pp. 20324-20336).

Clinical Applications

Integrins were found to be involved in pathological processes of both acute and chronic diseases such as ocular disease, cancer (primary tumors and metastasis), cardiovascular (stroke and heart failure) and inflammatory conditions (rheumatoid arthritis). Evidence clearly demonstrates that the αv integrin is associated with multiple tumor cells, including human breast, renal, cervical, colon, prostate, bladder, lung and melanoma. Antibodies to αv prevent human melanoma tumor formation in nude mice and antagonists of αvβ3 potentially inhibit angiogenesis in a number of animal models. Thus, blocking the αv integrin serves as an important therapeutic strategy in cancer therapy. Inhibitors of integrin function such as monoclonal antibody and peptide antagonist, which mimics the RGD ligand recognition domain common to αv integrin ligands, are now in phase II clinical trials.

ITAV-Splice Variant T5 Structure

The present inventors uncovered a novel isoform of integrin αV (ITAV; HUMVTNR_P5—SEQ ID NO:13, HUMVTNR_T5 (SEQ ID NO:15)—SEQ ID NO:15, FIGS. 12 a - b ) by applying LEADS clustering and assembly algorithm and the annotation process, as described above.

The ITAV splice variant T5 results from alternative splicing of the ITAV gene thereby introducing a novel exon, named exon 7a, between exons 7 and 8, leading to the insertion of a stop codon and the generation of a truncated ITAV protein ( FIGS. 12-15 ). ITAV splice variant T5 encodes a 298 amino acids long protein which contains the N-terminal signal sequence (residues 1-30), part of the extracellular domain of WT ITAV (SEQ ID NO:131) and a unique sequence of 45 amino acids at the C-terminus of the protein (ENTEALRRKITCPKSLACNLLFRDSNGDSLTPEVFFMMLNKSFGL SEQ ID NO:14). Since the ITAV splice variant of the present invention (SEQ ID NO:13) does not include the transmembrane domain present in the WT protein (amino acids 993-1016 of the WT protein, SEQ ID NO:131), it is predicated to be a secreted protein.

Comparison Report Between HUMVTNR_P5 (SEQ ID NO:13) and ITAV_HUMAN (SEQ ID NO:131)

1. An isolated chimeric polypeptide HUMVTNR_P5 (SEQ ID NO:13), comprising a first amino acid sequence being at least 90% homologous to MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFGFAV DPPVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPIEFDATG NRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGT CFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYW QGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLG corresponding to amino acids 1-253 of ITAV_HUMAN (SEQ ID NO:131), which also corresponds to amino acids 1-253 of HUMVTNR_P5 (SEQ ID NO:13), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ENTEALRRKITCPKSLACNLLFRDSNGDSLTPEVFFMMLNKSFGL (SEQ ID NO:14) corresponding to amino acids 254-298 of HUMVTNR_P5 (SEQ ID NO:13), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMVTNR_P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ENTEALRRKITCPKSLACNLLFRDSNGDSLTPEVFFMMLNKSFGL (SEQ ID NO:14) in HUMVTNR_P5 (SEQ ID NO:13).

Therapeutic Applications for the ITAV Splice Variant of the Present Invention

ITAV splice variant T3 could serve as a powerful antagonist of a variety of integrin interactions. It contains most of the extracellular region of ITAV and therefore is likely to bind the integrin αv ligands. This splice variant can inhibit the integrin signaling by competing with the membrane-bound receptor for the different ligands, thus preventing their binding to the cell surface receptor and as a consequence blocking integrin activation and signaling pathway. Alternatively, it can compete with the WT membrane ITAV for binding of the β subunit, thus preventing the heterodimerization of αv with the β subunit and the subsequent signaling.

Because of the overwhelming evidence favoring the role of αv integrin in the pathogenesis of a wide array of diseases as cancer, cardiovascular and inflammation, inhibitors of this molecule, such as ITAV splice variant T5 (SEQ ID NO:13), may have an important therapeutic potential. ITAV splice variant can play a crucial role in the treatment of the following pathological conditions: cancer (in general, but in particular colon and melanoma); cardiovascular diseases, such as atherosclerosis, restenosis, ischemia and reperfusion injury; immunological related diseases such as immunodeficiency, allergies, asthma, psoriasis, RA and inflammatory bowl diseases/chrone's disease; metabolism related diseases, such as diabetes and diabetes related retinopathy; osteoporosis, sepsis and wound healing.

Thus, the present inventors uncovered a therapeutic agent which can be used to: (i) prevent the binding of endogenous integrin αv with an integrin αv ligand (e.g., ECM proteins or cell surface proteins via e.g., the RGD sequence), (ii) prevent the binding of endogenous integrin αv with a β subunit (e.g., β1, β3, β5, β6, and β8) and thus prevent integrin αv-mediated cell signaling, (iii) treat a disorder associated with integrin αv-mediated cell signaling such as cancer (e.g., colon cancer and melanoma), cardiovascular diseases (e.g., atherosclerosis, restenosis, ischemia and reperfusion injury), immunological related diseases (e.g., immunodeficiency, allergies, asthma, psoriasis, rheumatoid arthritis (RA), inflammatory bowl disease, Crohn's disease, metabolism related diseases (e.g., diabetes and diabetes related retinopathy), osteoporosis, sepsis and wound healing by preventing the binding of the endogenous αv integrin with at least one αv ligand (e.g., ECM proteins or cell surface proteins via e.g., the RGD sequence) and/or β subunit (e.g., β1, β3, β5, β6, and β8). Such an agent is a polypeptide homologous to the integrin αv (ITAV) variant of the present invention (SEQ ID NO:13), and/or a polynucleotide homologous to SEQ ID NO: 15, and/or the unique peptide derived from the ITAV variant of the present invention (SEQ ID NO:14).

It will be appreciated that such an agent can be administered or provided to an individual in need thereof per se or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

Interferon-α/β-Receptor-1-INR1

Type I interferons (IFNs), initially identified for their ability to protect cells from viral infections, are truly pleiotropic cytokines. IFNs are implicated in both normal and neoplastic cell growth regulation and in modulating both innate and adaptive immune responses to microbial challenge. All type I IFNs, IFN-αs, IFN-β, IFN-ω, IFN-κ, and IFN-τ, are functionally active as monomers and activate a specific receptor complex composed of two major subunits, IFNAR-1/INR1 and IFNAR-2/INR2. The high affinity interaction between IFN-α/β and its specific cell surface receptor leads to receptor aggregation and the activation of receptor-associated cytoplasmic tyrosine kinases of the Jak family-Jak1 and Tyk2. These in turn phosphorylate intracellular tyrosine residues of the IFNAR-1 and IFNAR-2 chains, that serve as recruitment sites for the signal transducers and activators of transcription (STAT) proteins, Stat 1-5. Once associated with the activated receptor, the STAT become phosphorylated and form both homodimers and heterodimers, which translocate to the nucleus and bind specific DNA sequences within the promoter regions of IFN-sensitive genes (ISG). The Jak-Stat pathway is an essential signaling pathway for the transcription of many ISGs, whose protein products mediate specific IFN-dependent biologic responses. IFNs mediate a critical role in innate cellular defense against viral infection. Mice deficient in IFN-β or in IFNAR-1 are highly susceptible to viral infections. The antiviral activity of INFs include inhibition of viral replication and protein synthesis and the induction of viral mRNA degradation. In addition to their antiviral activity, IFNs exhibit growth inhibitory activity, either by mediating cell death (through caspases) or by modulating the expression of proteins regulating cell cycle entry and exit, hence mediating growth arrest. IFNs are also involved in the regulation of immune response towards viral or tumor challenge; A well-characterized function of IFNs is their ability to upregulate MHC class I expression and consequently promote CD8+ T cell responses. Moreover, IFNs can regulate the expression of key cytokines that influence T cell responses, namely, IL-12, IL-15 and IFN-γ and of CC-chemokines. IFNs-α/β regulate the functions of immune cells from different lineages including NK cells, dentritic cells and B/T lymphocytes (Deonarain et al. 2002. Current Pharmaceutical Design. Vol. 8, No. 24, Pp. 2131-2137; Brierley et al. 2002. Journal of Interferon and Cytokine Research. 22:835-845).

Clinical Application

Due to their growth inhibitory activity and the modulation of immune responses, type I interferons have been used as therapeutic polynucleotide or polypeptide sequences against a variety of solid tumors and hematological malignancies. IFN-α has been approved for the treatment of chronic myelogenous leukemia (CML), multiple myeloma, hairy cell leukemia and several lymphomas. Thus, IFN-α is the treatment of choice for CML patients which are not eligible for allogeneic bone marrow transplantation. In addition, the therapeutic efficacy of IFNs polynucleotide or polypeptide sequences in the treatment of viral infections and autoimmune diseases has been proved. Thus, IFN-α is the treatment of choice for hepatitis B and C infections and accumulating evidence supports the use of IFN-β for the treatment of multiple sclerosis.

However, although the activity and specificity of function make the IFNs potentially powerful therapeutic agents, they are not the ideal drugs, mainly due to their low stability in vivo.

Thus, there is an intense interest and effort to develop alternative or improved molecules demonstrating IFNs function with superior pharmacological properties. For example, PEGylation of type I IFNs extends the serum half-life and duration of therapeutic activity. PEGylation of IFN-α and IFN-β increased their serum half-life by 6 and 5 fold, respectively, however the PEGylated form of IFN-β exhibited less efficient systemic distribution with some evidence of induction of neutralizing antibodies. In addition, as opposed to their well-characterized function as competitive inhibitors (antagonists), soluble receptors have been shown to exhibit agonistic properties. These include increasing the molecular internal stability of the ligand, protection from proteolysis and modification of the pharmacokinetic properties of the ligand, namely, increasing the in vivo half-life of the ligand while decreasing its clearance. Thus, a soluble form of an IFN receptor should increase the in vivo stability of the ligand (i.e., IFN) and its agonistic properties.

INR1-Splice Variant Structure

The present inventors uncovered a novel isoform of interferon α/β receptor 1 (INR1) named INR1 splice variant T12 [T07758_P6—SEQ ID NO:17 ( FIG. 16B ); T07758_T12—SEQ ID NO:19 ( FIG. 16 a )]. INR1 splice variant T12 results from alternative splicing of the INR1 gene, thus introducing a novel exon, named exon 6a, between exons 6 and 7, leading to an insertion of a stop codon and the generation of a truncated protein. INR1 splice variant T12 (SEQ ID NO:19) encodes a 269 amino acids long protein (SEQ ID NO:17) containing the N-terminal signal sequence (residues 1-27), part of the extracellular portion of the WT INR1 (INR1_HUMAN SEQ ID NO:132), including the first two fibronectin type III-like domains and part of the third domain and a unique sequence of 7 amino acids (LYFRRPR—SEQ ID NO:18) at the C-terminus of the protein. Since the INR1 variant T12 (SEQ ID NO:17) does not include the transmembrane domain (which corresponds to residues 437-457 of WT INR1 SEQ ID NO:132), it is predicated to be a secreted protein.

Comparison Report Between T07758_P6 (SEQ ID NO:17) and INR1_HUMAN_V1 (SEQ ID NO:658)

1. An isolated chimeric polypeptide T07758_P6 (SEQ ID NO:17), comprising a first amino acid sequence being at least 90% homologous to MVVLLGATTLVLVAVAPWVLSAAAGGKNLKSPQKVEVDIIDDNFILRWNRS DESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIR AEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWAL DGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKI GVYSPVHClKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLH corresponding to amino acids 2-263 of INR1_HUMAN_V1, which also corresponds to amino acids 1-262 of T07758_P6 (SEQ ID NO:17), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LYFRRPR (SEQ ID NO:18) corresponding to amino acids 263-269 of T07758_P6 (SEQ ID NO:17), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of T07758_P6 (SEQ ID NO:17), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LYFRRPR (SEQ ID NO:18) in T07758_P6 (SEQ ID NO:17).

Comparison Report Between T07758_P6 (SEQ ID NO:17) and INR1_HUMAN (SEQ ID NO:132)

1. An isolated chimeric polypeptide T07758_P6 (SEQ ID NO:17), comprising a first amino acid sequence being at least 90% homologous to MVVLLGATTLVLVAV corresponding to amino acids 2-16 of INR1_HUMAN (SEQ ID NO:132), which also corresponds to amino acids 1-15 of T07758_P6 (SEQ ID NO:17), a bridging amino acid A corresponding to amino acid 16 of T07758_P6 (SEQ ID NO:17), a second amino acid sequence being at least 90% homologous to PWVLSAAAGGKNLKSPQKVEVDIIDDNFILRWNRSDESVONVTFSFDYQKTG MDNWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPF RKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLSFTYSL corresponding to amino acids 18-167 of INR1_HUMAN (SEQ ID NO:132), which also corresponds to amino acids 17-166 of T07758_P6 (SEQ ID NO:17), a bridging amino acid V corresponding to amino acid 167 of T07758_P6 (SEQ ID NO:17), a third amino acid sequence being at least 90% homologous to IWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIK TTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLH corresponding to amino acids 169-263 of INR1_HUMAN (SEQ ID NO:132), which also corresponds to amino acids 168-262 of T07758_P6 (SEQ ID NO:17), and a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LYFRRPR (SEQ ID NO:18) corresponding to amino acids 263-269 of T07758_P6 (SEQ ID NO:17), wherein said first amino acid sequence, bridging amino acid, second amino acid sequence, bridging amino acid, third amino acid sequence and fourth amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of T07758_P6 (SEQ ID NO:17), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LYFRRPR (SEQ ID NO:18) in T07758_P6 (SEQ ID NO:17).

Therapeutic Applications for the INR1 Splice Variant T12 of the Present Invention

The INR1 splice variant T12 of the present invention encodes a soluble receptor and can serve as an agonist of the INR1 by increasing the half-life of IFNs in vivo and enhancing their biological effect. Thus, the INR1 splice variant T12 (SEQ ID NO:17) may have an important therapeutic potential for the treatment of the following pathological conditions: cancer, such as solid tumors (e.g., glioblastoma, renal cell carcinoma, melanoma) and hematological malignancies (e.g., chronic myelogenous leukemia (CML), multiple myeloma, non-Hodgkin's lymphoma and hairy cell leukemia), viral infections (e.g., hepatitis B/C, herpes and human papilloma virus) and autoimmune diseases such as multiple sclerosis.

Thus, the present inventors uncovered a therapeutic agent which can be used to: (i) increase the in vivo stability of INF (e.g., IFN-α, IFN-β, IFN-ω, IFN-κ, and IFN-τ), (ii) increase INR1-mediated signaling by stabilizing the interaction between INF (e.g., IFN-α, IFN-β, IFN-ω, IFN-κ, and IFN-τ) and INR1, (iii) treat a disorder associated with INR1 signaling such as cancer [e.g., solid tumors (glioblastoma, renal cell carcinoma, melanoma) or hematological malignancies (chronic myelogenous leukemia (CML), multiple myeloma, non-Hodgkin's lymphoma and hairy cell leukemia)], viral infections (e.g., hepatitis B, hepatitis C, herpes and human papilloma virus) and autoimmune diseases such as multiple sclerosis, by stabilizing the INF (e.g., IFN-α, IFN-β, IFN-ω, IFN-κ, and IFN-τ) or the INF-INR1 interaction. Such an agent is a polypeptide homologous to the interferon a/13 receptor 1 (INR1) variant T12 of the present invention (SEQ ID NO:17), and/or a polynucleotide homologous to SEQ ID NO:19.

It will be appreciated that such an agent can be administered or provided to an individual in need thereof per se or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

Overexpression of a Troponin Variant in Cancer

The regulatory protein troponin (Tn) located on actin filament consists of three subunits: TnT, which binds troponin to tropomyosin, TnC, which binds divalent calcium ions, and TnI, which affects myosin-actin interactions. Tn subunits display several molecular and calcium binding variations. During ontogenetic development of cardiac and skeletal muscles the synthesis of multiple isoforms of Tn subunits was detected. Expression of Tn isoforms and the extent of phosphorylation of both TnT and TnI via protein kinase C or protein kinase A under different pathological situations (e.g. ischemia, congenital heart disease, heart failure) can affect the Ca 2+ -stimulated contraction function and the myofibrillar ATPase activity of the heart [Adamcova (1999) Physiol. Res. 48:235-2471. Troponin is commonly used as a marker for predicting cancer-therapy-induced cardiotoxicity. To date no reliable association has been made between cancer onset or progression and troponin expression.

By applying the teachings of the present invention, the present inventors uncovered elevated levels of novel troponin isoforms (see FIG. 24 and SEQ ID NOs:46-61) in lung, ovarian and colon cancers, suggesting the use of troponin alone or in combination with wild type troponin for diagnosis and treatment of cancer (see Examples 7a-c).

Materials and Experimental Procedures

RNA preparation—RNA was purchased from various sources including Clontech (Franklin Lakes, N.J. USA 07417), BioChain Inst. Inc., ABS or Ambion. Alternatively, RNA was purified from tissue samples using TRI-Reagent (Molecular Research Center), according to Manufacturer's instructions. Tissue samples were obtained from cancer patients or from postmortem. Total RNA samples were treated with DNaseI (Ambion) then purified using RNeasy columns (Qiagen).

RT PCR—1 μg of DNaseI-treated RNA was mixed with 150 ng of Random Hexamer primers (Invitrogen) and 500 μM dNTP in a total volume of 15.6 μl. The mixture was incubated for 5 min at 65° C. and then quickly chilled on ice. Thereafter, 5 μl of 5× SuperscriptII first strand buffer (Invitrogen), 2.4 μl of 0.1M DTT and 40 units RNasin (Promega) were added, and the mixture was incubated for 10 minutes at 25° C., followed by a 2-minutes at 42° C. Reverse transcription was effected by the addition of 1 μl (200 units) of SuperscriptII (Invitrogen) to the reaction mixture (final volume of 25 μl) and incubation at 42° C. for 50 minutes, following which the enzyme was inactivated at 70° C. for 15 minutes. The resulting cDNA was diluted 1:20 in TE (10 mM Tris pH=8, 1 mM EDTA pH=8).

Real-Time RT-PCR analysis—5 μl of diluted cDNA generated as described above were used as a template in Real-Time PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers (for example, SEQ ID NOs:42 and 43). UNG Enzyme (Eurogentech Cat. No. 2L, or ABI Cat. No. D12107 or Roche Cat. No. 10232921) was also included in the reactions. The gene-specific amplification was effected as follows: 50° C. for 2 minutes, 95° C. for 10 minutes, and then 40 cycles of 95° C. for 15 seconds, followed by 60° C. for 1 minute. Detection was effected using the SDS 7000 apparatus (PE Applied Biosystem). The cycle in which the reactions achieved a threshold level (Ct) of fluorescence was registered and served to calculate the relative transcript quantity in the RT reactions. The relative quantity was calculated using the following equation: Q=efficiency^-Ct. The efficiency of the PCR reaction was calculated from a standard curve created using serial dilutions of reverse transcription (RT) reactions prepared from RNA purified from 5 cell-lines (HCT116, H1299, DU145, MCF7, ES-2). To minimize inherent differences in the RT reaction, the resulting relative quantities were normalized to the geometric mean of the relative quantities of several housekeeping genes.

Detection of the expression level of troponin isoforms in normal, benign and cancerous ovary tissues—Expression of the troponin isoforms of the present invention (S69208_unique_region; SEQ ID NO:45) was measured by real time PCR using a fragment derived therefrom (amplicon—SEQ ID NO:23, primers are set forth in SEQ ID NOs:21 and 22). In addition the expression of four housekeeping genes—PBGD (GenBank Accession No. BC019323; amplicon—SEQ ID NO:32; Forward primer—SEQ ID NO:30; Reverse primer—SEQ ID NO:31), HPRT (GenBank Accession No. NM — 000194; amplicon—SEQ ID NO:29; Forward primer—SEQ ID NO:27; Reverse primer—SEQ ID NO:28), GAPDH (GenBank Accession No. BC026907; amplicon—SEQ ID NO:35; Forward primer—SEQ ID NO:33; Reverse primer—SEQ ID NO:34) and SDHA (GenBank Accession No. NM — 004168; amplicon—SEQ ID NO:26; Forward primer—SEQ ID NO:24; Reverse primer—SEQ ID NO:25) was measured by real time PCR. In each RT sample, the expression level of troponin-S69208_unique_region amplicon (SEQ ID NO:23) was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the averaged quantity of the normal post-mortem samples (no. 45-48, 71, Table 3, hereinbelow) to obtain a value of fold up-regulation of each sample relative to averaged normal samples.

Detection of the expression level of troponin isoforms in normal and cancerous lung tissues—was performed as described hereinabove for ovary tissues except that the expression level of Ubiquitin (GenBank Accession No. BC000449; amplicon—SEQ ID NO:38; forward primer—SEQ ID NO:36; reverse primer—SEQ ID NO:37) was determined instead of that of GAPDH and was used to normalize the expression level of the troponin-S69208_unique_region amplicon (SEQ ID NO:23). The normalized quantity of each RT sample was then divided by the averaged quantity of the normal post-mortem samples (no. 47-50, 90-93, 96-99, Table 4, hereinbelow) to obtain a value of fold up-regulation of each sample relative to averaged normal samples.

Detection of the expression level of troponin isoforms in normal and cancerous colon tissues—was performed as described hereinabove for ovary tissues except that the expression level of RPS27A (GenBank Accession No. NM — 002954; amplicon—SEQ ID NO:44; forward primer—SEQ ID NO:42; reverse primer—SEQ ID NO:43) and G6PD (GenBank Accession No. NM — 000402; amplicon—SEQ ID NO:41; forward primer—SEQ ID NO:39; reverse primer—SEQ ID NO:40) was determined instead of that of GAPDH and SDHA and was used to normalize the expression level of the troponin-S69208_unique_region amplicon (SEQ ID NO:23). The normalized quantity of each RT sample was then divided by the averaged quantity of the normal post-mortem samples (no. 41,52,62-67, 69-71 Table 5, hereinbelow) to obtain a value of fold up-regulation of each sample relative to averaged normal samples.

Experimental Results

Expression of troponin isoforms in normal, benign and cancerous ovary tissues—As shown in FIG. 25 , the expression of troponin-S69208_unique_region (SEQ ID NO:23) in normal samples (samples no. 45-52, 67-69, 71-75, Table 3, hereinbelow) and benign samples (samples 56-64, Table 2, hereinbelow) was significantly lower than in cancerous samples. Notably, troponin-S69208_unique_region up-regulation of at least 10 fold was found in 8 out of 15 adenocacinoma, 2 out of 7 Mucinus adenocarcinoma, 4 out of 9 Serous adenocarcinoma, 3 out of 5 mix serous-endometroid adenocarcinoma, 1 out of 3 endometroid adenocarcinoma, and in 2 of 2 clear-cell adenocarcinoma samples. A 10-15 fold up-regulation was observed also in 2 of the 11 matched normal samples. However, since matched samples are histologically non-cancerous tissue that surrounds the tumor, such samples could have been contaminated with cancer or pre-cancer cells.

Expression level of troponin isoforms in normal and cancerous lung tissues—As shown in FIG. 26 , the expression of troponin-S69208_unique_region (SEQ ID NO:23) was upregulated in several cancer samples relative to the normal samples. Specifically, troponin-S69208_unique_region up-regulation of at least 10 fold was found in 2 of 15 adenocarcinoma, 2 out of 16 squamous, 3 out of 4 large cell, and 2 out of 8 small cell samples. Notably, up-regulation of troponin-S69208_unique_region seems to be more specific to large cell tumors.

Expression of troponin isoforms in normal and cancerous colon tissues—As shown in FIG. 27 , the expression of troponin-S69208_unique_region was upregulated at least 10 fold (4 samples showed at least 5 fold) in two cancer samples relative to the normal samples. One of the 3 autoimmune disease samples also showed up-regulation in the expression of troponin S69208_unique_region.

Thus, these results demonstrate that troponin S69208_unique_region (SEQ ID NO:45) is upregulated in various cancers including lung, ovarian and colon cancers.

While reducing the present invention to practice, the present inventors have uncovered that the troponin variant of the present invention (SEQ ID NO:45), the PCR amplicon (SEQ ID NO:23) and/or the PCR primers (SEQ ID NO:21 and 22) used to detect such a variant, alone or in any combination thereof, can be used as diagnostic markers for diagnosing cancers (e.g., lung, ovarian and colon cancers). Detection of the expression level of the troponin variant of the present invention can be effected using methods immunological assays [e.g., Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence and the like, using an antibody directed against the troponin variant (SEQ ID NO:54), or by nucleic acid techniques (NAT) such as RT-PCR, Northern Blot, in situ hybridization, in situ RT-PCR.

While further reducing the present invention to practice, the present inventors have uncovered a method and pharmaceutical compositions for treating a troponin—related cancer (e.g., lung, ovarian and colon cancers). The method comprising downregulating the expression level and/or activity of at least one tropoinin variant, a polypeptide homologous to SEQ ID NO:54, to thereby treat the troponin—related cancer. Downregulating is effected using an agent selected from the group consisting of a troponin S69208_unique_region specific antisense oligonucleotide, a troponin S69208_unique_region specific siRNA, a troponin S69208_unique_region specific DNAzyme, a troponin S69208_unique_region specific Ribozyme, a troponin S69208_unique_region specific antibody, and a non-functional analogue of the troponin S69208_unique_region. Such an agent is provided at a therapeutic concentration along with a pharmaceutically acceptable carrier (e.g., PEG and liposomes).

Splice Variant of Integrin Alpha-M Precursor

Integrin αM associates in a non-covalent manner with β2 and generates the leukocyte membrane glycoprotein known as Mac-1, CR3, CD11b/CD18, or αMβ2-integrin. Mac-1 is one of four members of leukocyte-restricted β2-integrin family. It is expressed by granulocytes, monocytes, macrophages, dendritic cells, neutrophils eosenophils, natural killers (NKs) and some specific subsets of T and B lymphocytes. The expression and functional activity of Mac-1 is regulated during leukocyte differentiation and activation. Mac-1 is stored in intracellular vesicular compartments and is rapidly mobilized to the surface upon chemoattractants or other cellular stimuli. Mac-1 has two distinct functions; it mediates the migration of myeloid leukocytes and NKs out of blood vessels and into inflammatory sites by generating a high affinity binding site for intercellular adhesion molecule-1 (ICAM-1) expressed by activated endothelium. Additionally, as complement receptor type 3 (CR3), it mediates phagocytosis and cytotoxic degranulation in response to microorganisms or immune complexes opsonized with iC3b. It also forms complexes with other membrane glycoproteins, functioning as signal transducing partners for them. For this purpose, Mac-1 goes through series of “inside-out” and/or “outside-in” signaling steps that result in exposure of high affinity binding sites and/or an altered linkage to cytoskeletal elements (Ross, 2000, Critical Reviews in Immunology 20:197-222).

As is shown in FIG. 48 , the extracellular portion of Mac-1 encompasses seven (I-VII) homologues tandem repeats (FG-GAPS). Between FG-GAP II-III there is a von Willebrand sequence that contains the I-domain, which includes the binding site for all known protein ligands of Mac-1. A conformational shift that occurs upon binding a metal cation to MIDAS (metal-ion-dependent adhesion site) is responsible for the ligand binding activity of the I-domain (Takagi and Springer, 2002, Immunological Reviews 186:141-163). Domains V-VII contain putative divalent cation binding sites (also designated EF-hands). The seven FG-GAPS fold into a β propeller structure. The extracellular C-terminal region was found to contain a lectin-like site, which was shown to mediate both adhesion and cytotoxicity. Additionally Mac-1 has a transmembrane domain and a cytoplasmic tail.

Mac-1 recognizes a wide variety of ligands including collagen, fibronectin, heparan sulfate, ICAM-1, the complement component iC3b, Neutrophil inhibitory factor (NIF), factor X, and lipopolysaccharide (LPS) [Ross, 2000 (Supra)].

Clinical Applications

Increased expression of Mac-1 on circulating leukocytes occurs in several inflammatory disorders associated with neutrophil and monocyte activation e.g., in patients with burns, sepsis, hemodialysis, systemic lupus erythromatosis, diabetes mellitus, and in coronary artery disease. Neutrophil accumulation has been also demonstrated in ischemia reperfusion injury. Mac-1 deficiencies eliminate or markedly attenuate acute cellular inflammatory responses in vivo, suggesting that blocking Mac-1 activity may attenuate the tissue damage induced by cells overexpressing Mac-1. Thus, a mAb directed against the integrin αM subunit was found to be efficient in a dog model of myocardial reperfusion injury but only if administered well before reperfusion (Mazzone and Ricevuti, 1995, Haematologica 80:161-175). In addition, a recombinant NIF was tested in a phase II clinical trial as a possible therapeutic agent for the treatment of ischemia [Ross, 2000, (Supra); Takagi and Springer, 2002 (Supra); Mazzone and Ricevuti, 1995, (Supra); Zhou et al., 1994, JBC 269:17075-17079; Ueda et al., 1994, PNAS, 91: 10680-10684].

Splice Variant Structure

The present inventors uncovered a novel splice variant of Integrin alpha M Transcript T8 (HUMLAPA_T8—SEQ ID NO:78, HUMLAPA_P8—SEQ ID NO:77; FIGS. 45 a - b ). The T8 splice variant results from alternative splicing of the integrin αM gene, thus introducing an extension of exon 8 leading to the insertion of a stop codon and the generation of a truncated protein ( FIGS. 46-48 ). This splice variant (SEQ ID NO:78) encodes a 317 amino acids long protein (SEQ ID NO:77), containing 288 amino acids of the wild type sequence, and 29 unique amino acids (NAALRLMLLWRVSMWIHPPFNLQILLKSK—SEQ ID NO:79). It encompasses the FG-GAPS I and II and part of the von Willebrand domain, while lacking the FG-GAPs III-VII, the lectin domain (whose exact location is unknown), the TM and the cytoplasmic domain (see FIG. 48 ).

Comparison Report Between HUMLAPA_P8 and ITAM_HUMAN

1. An isolated chimeric polypeptide HUMLAPA_P8 (SEQ ID NO:77), comprising a first amino acid sequence being at least 90% homologous to MALRVLLLTALTLCHGFNLDTENAMTFQENARGFGQSVVQLQGSRVVVGAP QEIVAANQRGSLYQCDYSTGSCEPIRLQVPVEAVNMSLGLSLAATTSPPQLLA CGPTVHQTCSENTYVKGLCFLEGSNLRQQPQKFPEALRGCPQEDSDIAFLIDG SGSIIPHDFRRMKEFVSTVMEQLKKSKTLFSLMQYSEEFRIHFIFKEFQNNPNP RSLVKPITQLLGRTHTATGIRKVVRELFNITNGARKNAFKILVVITDGEKFGDP LGYEDVIPEADREGVIRYVIGVG corresponding to amino acids 1-288 of ITAM_HUMAN (SEQ ID NO:141), which also corresponds to amino acids 1-288 of HUMLAPA_P8 (SEQ ID NO:77), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NAALRLMLLWRVSMWIHPPFNLQILLKSK (SEQ ID NO:79) corresponding to amino acids 289-317 of HUMLAPA_P8 (SEQ ID NO:77), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMLAPA_P8, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NAALRLMLLWRVSMWIHPPFNLQILLKSK (SEQ ID NO:79) in HUMLAPA_P8 (SEQ ID NO:77).

Therapeutic Applications of the Variant

Three potential activities with therapeutic value could be attributed to a soluble αM molecule: (i) competing for dimerization with the β2 subunit and blocking the formation of an active αMβ2 Mac-1 integrin; (ii) competing with Mac-1 for ligand binding and blocking Mac-1-mediated adhesion; and (iii) competing with Mac-1 for binding the complement factor iC3b that mediates phagocytosis of opsonized tumor and bacterial cells.

Zhou et al. (1994, Supra) have demonstrated that recombinant soluble I-domain of αM binds fibrinogen and ICAM-1 (but not factor X) in vitro. The construct used in that work encompasses a larger region of the von Willebrand domain (Gly-127-Ala-334) that is included in the T8 variant of the present invention (SEQ ID NO:77). In addition, Ueda and colleagues (1994, Supra) have shown that immobilized recombinant (rCD11b) I-domain of CD11b is capable of binding complement component-coated erythrocytes (EAiC3b I-domain) in a dose-dependent manner and that such binding is inhibited by soluble rCD11b I-domain. Furthermore, they have shown that a short, linear, I domain peptide (residues 232-245) (i) binds the ligand in a dose dependent manner; (ii) inhibits ligand binding to immobilized rCD11b I-domain, (iii) inhibits binding of erythrocytes to rCD11b I-domain; and (iv) inhibits binding of erythrocytes to neutrophils.

Altogether, these data support a role for T8 (SEQ ID NO:77) as an antagonist of Mac-1 ligand binding and of Mac-1-dependent complement activation.

Thus, the present inventors uncovered a therapeutic agent which can be used to: (i) prevent the dimerization of endogenous αM integrin (SEQ ID NO:141) with β2 (SEQ ID NO:165) and thus block the formation of an active αMβ2 Mac-1 integrin, (ii) prevent the binding of a Mac-1 ligand [e.g., collagen, fibronectin, heparan sulfate, ICAM-1, the complement component iC3b, Neutrophil inhibitory factor (NIF), factor X, and lipopolysaccharide (LPS)] with an endogenous Mac-1 and thus block Mac-1-mediated adhesion (iii) prevent binding of endogenous Mac-1 with the complement factor iC3b and thus prevent Mac-1-mediated phagocytosis of opsonized tumor and bacterial cells, (iv) prevent and/or treat a disorder associated with increased expression of Mac-1 (e.g., increased expression on circulating leukocytes). Such an agent is a polypeptide homologous to the αM variant T8 of the present invention (SEQ ID NO:77), and/or a polynucleotide homologous to SEQ ID NO:78 and/or the peptide derived from the αM variant T8 (SEQ ID NO:79). Such an agent can prevent the binding of a Mac-1 ligand [e.g., collagen, fibronectin, heparan sulfate, ICAM-1, the complement component iC3b, Neutrophil inhibitory factor (NIF), factor X, and lipopolysaccharide (LPS)] or an endogenous β2 (SEQ ID NO:165) with the endogenous αM (SEQ ID NO:141). Non-limiting examples of disorders which can be treated according to this aspect of the present invention include burns, sepsis, hemodialysis, systemic lupus erythromatosis, diabetes mellitus, in coronary artery disease, and ischemia reperfusion injury.

It will be appreciated that such an agent can be administered or provided to an individual in need thereof per se or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

Splice Variant of Interferon-Alpha/Beta Receptor Beta Chain Precursor

Type I interferons (IFNs; e.g., IFN-α, IFN-β, IFN-ω, IFN-κ, and IFN-τ) are implicated in both normal and neoplastic cell growth regulation and in modulating both innate and adaptive immune responses to microbial challenge. Thus ISNs exhibit antiviral, antiproliferative, immunomodulatory and developmental activities. All type I IFNs are functionally active as monomers and activate a specific receptor complex composed of two major subunits, IFNAR-1/INR1 and IFNAR-2/INR2. The high affinity interaction between IFN-α/β and its specific cell surface receptor leads to receptor aggregation and the activation of receptor-associated cytoplasmic tyrosine kinases of the Jak family-Jak1 and Tyk2. These in turn phosphorylate intracellular tyrosine residues of the IFNAR-1 and IFNAR-2 chains, that serve as recruitment sites for the signal transducers and activators of transcription (STAT) proteins, Stat 1-5. Once associated with the activated receptor, the STAT become phosphorylated and form both homodimers and heterodimers, which translocate to the nucleus and bind specific DNA sequences within the promoter regions of IFN-sensitive genes (ISG). The Jak-Stat pathway is an essential signaling pathway for the transcription of many ISGs, whose protein products mediate specific IFN-dependent biologic responses. IFNs mediate a critical role in innate cellular defense against viral infection. The antiviral activity of INFs include inhibition of viral replication and protein synthesis and the induction of viral mRNA degradation. In addition to their antiviral activity, IFNs exhibit growth inhibitory activity, either by mediating cell death (through caspases) or by modulating the expression of proteins regulating cell cycle entry and exit, hence mediating growth arrest. IFNs are also involved in the regulation of immune response towards viral or tumor challenge; A well-characterized function of IFNs is their ability to upregulate MHC class I expression and consequently promote CD8+ T cell responses. Moreover, IFNs can regulate the expression of key cytokines that influence T cell responses, namely, IL-12, IL-15 and IFN-γ and of CC-chemokines. IFNs-α/β regulate the functions of immune cells from different lineages including NK cells, dentritic cells and B/T lymphocytes (Deonarain et al. 2002. Current Pharmaceutical Design. Vol. 8, No. 24, Pp. 2131-2137; Brierley et al. 2002. Journal of Interferon and Cytokine Research. 22:835-845).

Prior attempts to inhibit the interferon mediated activities included the use of mono clonal antibodies directed against the IFNAR-2 receptor. These receptors neutralized type I IFN-mediated antiviral, antiproliferative, and major histocompatibility complex (MHC) class I upregulation functions (Novick D, et al., 2000; J. Interferon Cytokine Res. 20: 971-82).

Clinical Application

Due to their growth inhibitory activity and the modulation of immune responses, type I interferons have been used as therapeutic polynucleotide or polypeptide sequences against a variety of solid tumors and hematological malignancies. IFN-α has been approved for the treatment of chronic myelogenous leukemia (CML), multiple myeloma, hairy cell leukemia and several lymphomas. Thus, IFN-α is the treatment of choice for CML patients which are not eligible for allogeneic bone marrow transplantation. In addition, the therapeutic efficacy of IFNs polynucleotide or polypeptide sequences in the treatment of viral infections and autoimmune diseases has been proved. Thus, IFN-α is the treatment of choice for hepatitis B and C infections and accumulating evidence supports the use of IFN-β for the treatment of multiple sclerosis.

In addition, since INR2 is overexpressed in lymph nodes-tumors it can be used as a marker for lymph node tumors.

Splice Variant HSIFNABR_T14 (Transcript) Encodes a Secreted Form of INR2 (HSIFNABR_P8)

The present inventors have uncovered a new INR2 variant [HSIFNABR_P8-SEQ ID NO:155 ( FIG. 96 b ); HSIFNABR_T14—SEQ ID NO:156 ( FIG. 96 a )]. The protein coordinates on the transcript start from nucleotide 361 and end at nucleotide 951 as set forth in SEQ ID NO:156 (HSIFNABR_T14 transcript).

Alignment of the new INR2 variant (HSIFNABR_P8—SEQ ID NO:155) with the WT protein (GenBank Accession No. P48551; SEQ ID NO:157) revealed that the new variant includes the first 180 amino acids as of the WT protein (GenBank Accession No. P48551) followed by a unique 17 amino acid sequence RGEDEKLDISQFCHRQAL (SEQ ID NO:158), FIG. 97 ]. The new variant uncovered by the present invention lacks the Cytokine receptor class 2 (IPR000282) and the transmembrane domain of the WT protein (amino acids 244-264 of GenBank Accession No. P48551) and therefore is expected to be a secreted or a soluble protein.

Comparison Report Between HSIFNABR_P8 and INR2_HUMAN

1. An isolated chimeric polypeptide HSIFNABR_P8, comprising a first amino acid sequence being at least 90% homologous to MLLSQNAFIPRSLNLVLMVYISLVFGISYDSPDYTDESCTFKISLRNFRSILSWE LKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYV TVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEEL QFDLSLVIEEQSEGIVKK corresponding to amino acids 1-180 of INR2_HUMAN (SEQ ID NO:157), which also corresponds to amino acids 1-180 of HSIFNABR_P8 (SEQ ID NO:155), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GEDEKLDISQFCHRQAL (SEQ ID NO:158) corresponding to amino acids 181-197 of HSIFNABR_P8 (SEQ ID NO:155), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HSIFNABR_P8 (SEQ ID NO:155), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GEDEKLDISQFCHRQAL (SEQ ID NO:158) in HSIFNABR_P8 (SEQ ID NO:155).

Thus, the present inventors uncovered a therapeutic agent which can be used to: (i) increase the in vivo stability of INF (e.g., IFN-α, IFN-β, IFN-ω, IFN-κ, and IFN-τ), (ii) increase INR2-mediated signaling by stabilizing the interaction between INF (e.g., IFN-α, IFN-β, IFN-ω, IFN-κ, and IFN-τ) and INR2, (iii) treat a disorder associated with INR2 signaling such as cancer (e.g., leukaemia, chronic myelogenous, hairy cell cancer, lymphoma, non-Hodgkin's lymphoma, melanoma, myeloma, renal cancer, bone cancer, sarcoma, Kaposi's sarcoma, brain cancer, cervical cancer, head and neck cancer, skin cancer), infection [e.g., HIV/AIDS infection, coronavirus infection, prophylaxis infection, general infection, hepatitis virus infection (such as hepatitis-B, hepatitis-C), herpes simplex virus, herpes virus, human papilloma virus, varicella zoster virus], multiple sclerosis, Pemphigus, Behcet's disease, chronic fatigue syndrome, hepatic cirrhosis, fibromyalgia, pulmonary fibrosis, inflammation (brain), Keratoconjunctivitis, and macular degeneration. Such an agent is a polypeptide homologous to the INR2 variant of the present invention (HSIFNABR_P8) (SEQ ID NO: 155), and/or a polynucleotide homologous to SEQ ID NO:156, and/or a peptide homologous to GEDEKLDISQFCHRQAL (SEQ ID NO:158). It will be appreciated that such an agent can be administered per se or as part of a pharmaceutical composition along with a suitable pharmaceutical acceptable carrier (e.g., PEG and liposomes).

Thus, the present invention provides therapeutic agents which can be used as anticancer, antifungal, antiviral, anti-HIV, anti-AIDS, immunostimulant, immunomodulator, hepatoprotective, antiinfective agents, as well as for the treatment of Multiple sclerosis, musculoskeletal, neurological, ophthalmological, respiratory and stomatological diseases.

While further reducing the present invention to practice, the present inventors have uncovered that the INR2 variant of the present invention i.e., HSIFNABR_P8 (SEQ ID NO:155), HSIFNABR_T14 (SEQ ID NO:156) or the peptide derived therefrom [GEDEKLDISQFCHRQAL (SEQ ID NO:158)] can be used as a diagnostic marker for various cancers such as lymph node tumors.

The tumor necrosis factor receptor superfamily member 14 (TR14_HUMAN; GenBank Accession No. Q92956; SEQ ID NO:161; Type I membrane protein; Herpesvirus entry mediator A; Tumor necrosis factor receptor-like 2; TR2; The HUGO gene symbol of this product: HVEA; HVEM; TNFRSF14) is a cellular receptor for TNF superfamily 14 (LIGHT) which involves in immune response, cell surface receptor linked signal transduction and apoptosis. TR14 is overexpressed in skin and can be used as a marker for proliferation of this tissue or as a marker for pathological de-differentiation of this tissue or tissue damage. In addition, since TR14 is overexpressed in skin and pancreas tumors it can be used as a marker for these pathologies.

Splice Variant Z42185_T13 (SEQ ID NO:159) Encodes a New Secreted form of the TR14 Receptor, Z42185_P5 (SEQ ID NO:160)

The present inventors have uncovered a new TR14 variant V42185_P5—SEQ

ID NO:160 ( FIG. 98 b ); Z42185_T13—SEQ ID NO:159 ( FIG. 98 a )]. The protein coordinates on the transcript start from nucleotide 891 and end at nucleotide 1481 as set forth in SEQ ID NO:159 (Z42185_T13 transcript).

Alignment of the new TR14 variant (Z42185_P5—SEQ ID NO:160) with the WT protein (GenBank Accession No. Q92956; SEQ ID NO:161) revealed that the new variant includes the first 183 amino acids as of the WT protein (GenBank Accession No. Q92956) followed by a unique 14 amino acid sequence [(NWPNHMCEKKKAKG (SEQ ID NO:162), FIG. 99 ]. The new variant uncovered by the present invention lacks the transmembrane domain of the WT protein (amino acids 203-223 of GenBank Accession No. Q92956) and therefore is expected to be a secreted, soluble protein (i.e., extracellular).

Comparison Report Between Z42185_P5 (SEQ ID NO:160) and TR14_HUMAN (SEQ ID NO:161)

1. An isolated chimeric polypeptide Z42185_P5 (SEQ ID NO:160), comprising a first amino acid sequence being at least 90% homologous to MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPVGSE CCPKCSPGYRVKEACGELTGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMG LRASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPGQRVQKGGTE SQDTLCQNCPPGTFSPNGTLEECQHQT corresponding to amino acids 1-183 of TR14_HUMAN (SEQ ID NO:161), which also corresponds to amino acids 1-183 of Z42185_P5 (SEQ ID NO:160), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NWPNHMCEKKKAKG (SEQ ID NO:162) corresponding to amino acids 184-197 of Z42185_P5 (SEQ ID NO:160), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of Z42185_P5 (SEQ ID NO:160), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NWPNHMCEKKKAKG (SEQ ID NO:162) in Z42185_P5 (SEQ ID NO:160).

These results suggest the use of the new TR14 variant of the present invention (Z42185_P5—SEQ ID NO:160), the polynucleotide encoding same (Z42185_T13—SEQ ID NO:159) and/or the peptide derived from the Z42185_P5 TR14 variant (NWPNHMCEKKKAKG—SEQ ID NO:162) as a diagnostic marker for skin proliferation or de-differentiation, as well as skin and pancreas tumors. Diagnosis according to this aspect of the present invention is effected using immunological assays [e.g., Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence, and the like using an antibody directed against the TR14 variant (Z42185_P5—SEQ ID NO:160)], nucleic acid techniques (NAT) such as RT-PCR, Northern Blot, in situ hybridization, in situ RT-PCR.

Splice Variant of Integrin Beta-2 Precursor

The β2 integrin receptors (e.g., LFA-1, Mac-1, and p150,95) are expressed on leukocytes and play a major role in leukocyte cell-cell and cell-matrix adhesions during inflammation and other immune responses.

The integrin beta-2 precursor (Cell surface adhesion glycoproteins LFA-1/CR3/p150,95 beta-subunit; CD18; Complement receptor C3 beta-subunit; GenBank Accession No. P05107; ITB2_HUMAN; ITGB2) associates with an integrin α subunit (α-L, α-M, α-X, α-D) to form a Type I membrane protein receptor. Thus, integrin α-L/β-2 is a receptor for ICAM1, ICAM2, ICAM3 and ICAM4; integrins α-M/β-2 and α-X/β-2 are receptors for the iC3b fragment of the third complement component and for fibrinogen; integrin α-M/β-2 is also a receptor for factor X; integrin α-D/β-2 is a receptor for ICAM3 and VCAM1. The integrin receptors recognize specific ligands via recognition sequences such as the G-P—R in fibrinogen alpha-chain which is recognized by integrin α-X/β-2 and the P1 and P2 peptides of fibrinogen gamma chain which is recognized by integrin α-M/β-2.

Defects in ITGB2 are the cause of leukocyte adhesion deficiency type I (LAD1) [MIM:116920]. LAD1 patients have recurrent bacterial infections and their leukocytes are deficient in a wide range of adhesion-dependent functions. The integrin β-2 precursor protein contains one VWFA-like domain.

Clinical Applications

Since β2 integrin receptors are expressed on leukocytes and are involved in various cell-cell and cell-matrix adhesions during inflammation and other immune responses, inhibition of β2 integrins or the introducing of soluble forms of β2 integrins can be used as anti-inflammatory agents for the treatment of various diseases including cancers (e.g., breast cancer), coronary artery bypass grafting, haemorrhage, myocardial infarction, inflammation (e.g., pulmonary inflammation), cerebral ischaemia, osteoporosis, reperfusion injury, transplant rejection (e.g., bone marrow transplant rejection) and hepatic dysfunction.

For example, prior studies have shown that murine monoclonal antibodies directed against the CD11b/CD18 (CR3) heterodimer are capable of reducing the phagocyte-mediated ischemia-reperfusion injury in several organ systems, such as the myocardium, liver, and gastrointestinal tract, as well as inhibiting the development of insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice (Dana N, et al., 1991; Proc. Natl. Acad. Sci. USA, 88: 3106-10).

Splice Variant HUMLAP_T18 (SEQ ID NO:163) Encodes a New Secreted Form of Integrin β2, HUMLAP_P15 (SEQ ID NO:164)

The present inventors have uncovered a new integrin β2 variant [HUMLAP_P15—SEQ ID NO:164 ( FIG. 100 b ); HUMLAP_T18SEQ ID NO:163 ( FIG. 100 a )]. The protein coordinates on the transcript start from nucleotide 414 and end at nucleotide 737 as set forth in SEQ ID NO:163 (HUMLAP_T18 transcript).

Alignment of the new integrin β2 variant (HUMLAP_P15—SEQ ID NO:164) with the WT protein (GenBank Accession No. P05107; SEQ ID NO:165) revealed that the new variant includes the first 49 amino acids as of the WT protein (GenBank Accession No. P05107) followed by a unique 59 amino acid sequence [G AALGPPAHATAASSPRRRSRVAPVCPRTEQGGQAPGGNYLGQAGFFPSPFWR FSAPLK (SEQ ID NO:166), FIG. 101 ]. The new variant uncovered by the present invention lacks the majority of the ITGB2 mature sequence (IPR002369 Integrin, beta chain IPR003659 Plexin/semaphorin/integrin) including potential sites of glycosylation and the transmembrane domain of the WT protein (amino acids 701-723 of GenBank Accession No. P05107) and therefore is expected to be a secreted, soluble and extracellular protein.

Comparison Report Between HUMLAP_P15 and ITB2_HUMAN

1. An isolated chimeric polypeptide HUMLAP_P15, comprising a first amino acid sequence being at least 90% homologous to MLGLRPPLLALVGLLSLGCVLSQECTKFKVSSCRECIESGPGCTWCQKL corresponding to amino acids 1-49 of ITB2_HUMAN (SEQ ID NO:165), which also corresponds to amino acids 1-49 of HUMLAP_P15 (SEQ ID NO:164), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GAALGPPAHATAASSPRRRSRVAPVCPRTEQGGQAPGGNYLGQAGFFPSPFW RFSAPLK (SEQ ID NO:166) corresponding to amino acids 50-108 of HUMLAP_P15 (SEQ ID NO:164), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMLAP_P15 (SEQ ID NO:164), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GAALGPPAHATAASSPRRRSRVAPVCPRTEQGGQAPGGNYLGQAGFFPSPFW RFSAPLK (SEQ ID NO:166) in HUMLAP_P15 (SEQ ID NO:164).

Clinical Applications of Using the Integrin β2 Variant of the Present Invention

Since the integrin variant of the present invention, HUMLAP_P15 (SEQ ID NO:164), is a soluble extracellular protein it can be used as an integrin β2 antagonist and/or an anti-inflammatory agent in the treatment of various diseases.

Thus, the present inventors uncovered a therapeutic agent which can be used to treat an integrin β2-related disease or condition [e.g., various cancers such as breast cancer, cardiovascular disease, coronary artery bypass grafting, haemorrhage, myocardial infraction, inflammation (e.g., pulmonary inflammation, asthma, GI inflammation, bowel disorder), cerebral ischaemia, osteoporosis, reperfusion injury, transplant rejection (e.g., bone marrow transplant rejection), psoriasis, osteoporosis treatment, respiratory disease, and hepatic dysfunction. Such an agent is a polypeptide homologous to the integrin β2 variant of the present invention (SEQ ID NO:164), and/or a polynucleotide homologous to SEQ ID NO:163, and/or a peptide homologous to SEQ ID NO:166. It will be appreciated that the polypeptide, polynucleotide and/or peptide used according to this aspect of the present can be administered or provided per se, or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

While further reducing the present invention to practice, the present inventors have uncovered that the integrin β2 variant of the present invention HUMLAP_P15 (SEQ ID NO:164), the peptide derived therefrom (SEQ ID NO:166) and/or the polynucleotide encoding same (SEQ ID NO:163), each and in any combination, can be used as diagnostic markers for various cancers including leukemia (blood malignancies) and muscle tumors. Diagnosis according to this aspect of the present invention is effected using immunological assays [e.g., Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence, and the like using an antibody directed against the integrin β2 variant (SEQ ID NO:164), or by nucleic acid techniques (NAT) such as RT-PCR, Northern Blot, in situ hybridization, in situ RT-PCR.

Splice Variant of Integrin Beta-2 Precursor

The β2 integrin receptors (e.g., LFA-1, Mac-1, and p150,95) are expressed on leukocytes and play a major role in leukocyte cell-cell and cell-matrix adhesions during inflammation and other immune responses.

The integrin beta-2 precursor (Cell surface adhesion glycoproteins LFA-1/CR3/p150,95 beta-subunit; CD18; Complement receptor C3 beta-subunit; GenBank Accession No. P05107; ITB2_HUMAN; ITGB2) associates with an integrin α subunit (α-L, α-M, α-X, α-D) to form a Type I membrane protein receptor. Thus, integrin α-L/β-2 is a receptor for ICAM1, ICAM2, ICAM3 and ICAM4; integrins α-M/β-2 and α-X/β-2 are receptors for the iC3b fragment of the third complement component and for fibrinogen; integrin α-M/β-2 is also a receptor for factor X; integrin α-D/β-2 is a receptor for ICAM3 and VCAM1. The integrin receptors recognize specific ligands via recognition sequences such as the G-P—R in fibrinogen alpha-chain which is recognized by integrin α-X/β-2 and the P1 and P2 peptides of fibrinogen gamma chain which is recognized by integrin α-M/β-2.

Defects in ITGB2 are the cause of leukocyte adhesion deficiency type I (LAD1) [MIM:116920]. LAD1 patients have recurrent bacterial infections and their leukocytes are deficient in a wide range of adhesion-dependent functions. The integrin β-2 precursor protein contains one VWFA-like domain.

Clinical Applications

Since β2 integrin receptors are expressed on leukocytes and are involved in various cell-cell and cell-matrix adhesions during inflammation and other immune responses, inhibition of β2 integrins or the introducing of soluble forms of β2 integrins can be used as anti-inflammatory agents for the treatment of various diseases including cancers (e.g., breast cancer), coronary artery bypass grafting, haemorrhage, myocardial infarction, inflammation (e.g., pulmonary inflammation), cerebral ischaemia, osteoporosis, reperfusion injury, transplant rejection (e.g., bone marrow transplant rejection) and hepatic dysfunction.

For example, prior studies have shown that murine monoclonal antibodies directed against the CD11b/CD18 (CR3) heterodimer are capable of reducing the phagocyte-mediated ischemia-reperfusion injury in several organ systems, such as the myocardium, liver, and gastrointestinal tract, as well as inhibiting the development of insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice (Dana N, et al., 1991; Proc. Natl. Acad. Sci. USA, 88: 3106-10).

Splice Variant HUMLAP_T14 (SEQ ID NO:167) Encodes a New Secreted Form of Integrin β2, HUMLAP_P12 (SEQ ID NO:168)

The present inventors have uncovered a new integrin β2 variant [HUMLAP_P12 (SEQ ID NO:168); HUMLAP_T14 (SEQ ID NO:167)]. The protein coordinates on the transcript start from nucleotide 414 and end at nucleotide 1229 as set forth in SEQ ID NO:167 (HUMLAP_T14 transcript).

Alignment of the new integrin β2 variant (HUMLAP_P12—SEQ ID NO:168) with the WT protein (GenBank Accession No. P05107; SEQ ID NO:165) revealed that the new variant includes the first 217 amino acids as of the WT protein (GenBank Accession No. P05107) followed by a unique 55 amino acid sequence [SALKMTAMAGRVLLGARRGDSSTLTGTVFAWRLEEGGLEVGEVRCVFPVQ VRTSV (SEQ ID NO:169), FIG. 102 ]. The new variant uncovered by the present invention lacks the majority of the ITGB2 mature sequence (IPR002369 Integrin, beta chain), exhibits a truncated VWFA-like domain (amino acids 124-363 of GenBank Accession No. P05107) and lacks the transmembrane domain of the WT protein (amino acids 701-723 of GenBank Accession No. P05107) and therefore is expected to be a secreted, soluble and extracellular protein.

Comparison Report Between HUMLAP_P12 and ITB2_HUMAN

1. An isolated chimeric polypeptide HUMLAP_P12, comprising a first amino acid sequence being at least 90% homologous to MLGLRPPLLALVGLLSLGCVLSQECTKFKVSSCRECIESGPGCTWCQKLNFTG PGDPDSIRCDTRPQLLMRGCAADDIMDPTSLAETQEDHNGGQKQLSPQKVTL YLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYSMLDDLRNVKKLGGDLLRA LNEITESGRIGFGSFVDKTVLPFVNTHPDKLRNPCPNKEKECQPPFAFRHVLKL TNNSNQF corresponding to amino acids 1-217 of ITB2_HUMAN (SEQ ID NO:165), which also corresponds to amino acids 1-217 of HUMLAP_P12 (SEQ ID NO:168), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 90% homologous to a polypeptide having the sequence SALKMTAMAGRVLLGARRGDSSTLTGTVFAWRLEEGGLEVGEVRCVFPVQV RTSV (SEQ ID NO:169) corresponding to amino acids 218-272 of HUMLAP_P12 (SEQ ID NO:168), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMLAP_P12 (SEQ ID NO:168), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SALKMTAMAGRVLLGARRGDSSTLTGTVFAWRLEEGGLEVGEVRCVFPVQV RTSV (SEQ ID NO:169) in HUMLAP_P12 (SEQ ID NO:168).

Clinical Applications of Using the Integrin β2 Variant of the Present Invention

Since the integrin variant of the present invention, HUMLAP_P12 (SEQ ID NO:168), is a soluble extracellular protein it can be used as an integrin β2 antagonist and/or an anti-inflammatory agent in the treatment of various diseases.

Thus, the present inventors uncovered a therapeutic agent which can be used to treat an integrin β2-related disease or condition [e.g., various cancers such as breast cancer, cardiovascular disease, coronary artery bypass grafting, haemorrhage, myocardial infraction, inflammation (e.g., pulmonary inflammation, asthma, GI inflammation, bowel disorder), cerebral ischaemia, osteoporosis, reperfusion injury, transplant rejection (e.g., bone marrow transplant rejection), psoriasis, osteoporosis treatment, respiratory disease, and hepatic dysfunction. Such an agent is a polypeptide homologous to the integrin β2 variant of the present invention (SEQ ID NO:168), and/or a polynucleotide homologous to SEQ ID NO:167, and/or a peptide homologous to SEQ ID NO:169. It will be appreciated that the polypeptide, polynucleotide and/or peptide used according to this aspect of the present can be administered or provided per se, or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

While further reducing the present invention to practice, the present inventors have uncovered that the integrin β2 variant of the present invention HUMLAP_P12 (SEQ ID NO:168), the peptide derived therefrom (SEQ ID NO:169) and/or the polynucleotide encoding same (SEQ ID NO:167), each and in any combination, can be used as diagnostic markers for various cancers including leukemia (blood malignancies) and muscle tumors.

Diagnosis according to this aspect of the present invention is effected using immunological assays [e.g., Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence, and the like using an antibody directed against the integrin β2 variant (SEQ ID NO:168), or by nucleic acid techniques (NAT) such as RT-PCR, Northern Blot, in situ hybridization, in situ RT-PCR.

Splice Variant of Low Affinity Immunoglobulin Gamma Fc Region Receptor III-A Precursor

An essential element in the regulation of immune effector responses is via the antibody Fc receptors (FcRs) which are expressed or displayed in immune effectors. Fc receptors belong to the immunoglobulin superfamily, and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC) [Van de Winkel and Anderson, J. Leuk. Biol. 49:511-24 (1991)]. One subclass of Fc receptors includes the Fc gamma receptors, which are specific for the IgG antibodies. These include the FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). FcγRIII is a low-affinity receptor interacting with complexed or aggregated IgG.

The Fc region receptor III-A precursor Low affinity immunoglobulin gamma Fc region receptor III-A precursor (IgG FC receptor III-2; Fc-gamma RIII-alpha; FcRIIIA; CD16-A; FcR-10), is a type-I membrane protein, which, following proteolytic cleavage can exist as a soluble receptor [e.g., neutrophil-derived soluble FcRIII (S-FcRIII)]. It is expressed on natural killer cells, macrophages, mast cells, subpopulation of T cells, immature thymocytes and placental trophoblasts. The protein contains an extracellular domain (amino acids 17-208), Ig-like C2 type 1 and 2 domains (amino acids 24-105 and 107-189, respectively), five potential glycosylation sites (amino acids 56, 63, 92, 180 and 187), two potential disulfide bonds (amino acids 47, 89; and 128, 172), a transmembrane domain (amino acids 209-229) and a potential cytoplasmic domain (amino acids 230-254); all amino acids positions relate to GenBank Accession No. P08637; SEQ ID NO:173).

Clinical Applications

The FcγRII-A exhibits an important role in the immune response. It may be involved in various diseases or conditions such as allergy reactions, anaemia, rheumatoid arthritis, asthma, inflammation, lupus erythematosus, thrombocytopenia, and thrombocytopenic purpura. In addition, abnormal accumulation of CD16+ cells was associated with large granular lymphocyte proliferative disease (Blancho G, et al., 1992, Transplantation, 53(6):1242-7) and the kidney allograft failure.

Splice Variant HUMGCRFC_T4 (SEQ ID NO:170) Encodes a New Secreted Form of FCγRIII-A, HUMGCRFC_P3 (SEQ ID NO:171)

The present inventors have uncovered a new FCγRIII-A variant [HUMGCRFC_P3 (SEQ ID NO:171); HUMGCRFC_T4 (SEQ ID NO:170)]. The protein coordinates on the transcript start from nucleotide 106 and end at nucleotide 465 as set forth in SEQ ID NO:170 (HUMGCRFC_T4 transcript).

Alignment of the new FCγRIII-A variant (HUMGCRFC_P3—SEQ ID NO:171) with the WT protein (GenBank Accession No.; SEQ ID NO:173) revealed that the new variant includes the first 107 amino acids as of the WT protein (GenBank Accession No. P08637) followed by a unique 13 amino acid sequence [ELMKGKRKITNKG (SEQ ID NO:172), FIG. 103 ]. The new variant uncovered by the present invention lacks the Immunoglobulin/major histocompatibility complex (IPR003006) and the Immunoglobulin subtype (IPR003599) and therefore is expected to be a secreted, soluble and extracellular protein.

Comparison Report Between HUMGCRFC_P3 and FC3A_HUMAN

1. An isolated chimeric polypeptide HUMGCRFC_P3 (SEQ ID NO:171), comprising a first amino acid sequence being at least 90% homologous to MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAY SPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEV HIG corresponding to amino acids 1-107 of FC3A_HUMAN (SEQ ID NO:173), which also corresponds to amino acids 1-107 of HUMGCRFC_P3 (SEQ ID NO:171), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence ELMKGKRKITNKG (SEQ ID NO:172) corresponding to amino acids 108-120 of HUMGCRFC_P3 (SEQ ID NO:171), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMGCRFC_P3 (SEQ ID NO:171), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence ELMKGKRKITNKG (SEQ ID NO:172) in HUMGCRFC_P3 (SEQ ID NO:171).

Splice Variant HUMGCRFC_T5 (SEQ ID NO:174) Encodes a New Secreted Form of the FCγRIII-A, HUMGCRFC_P4 (SEQ ID NO:175)

The present inventors have uncovered a new FCγRIII-A variant [HUMGCRFC_T5—SEQ ID NO:174; HUMGCRFC_P4—SEQ ID NO:1751. The protein coordinates on the transcript start from nucleotide 106 and end at nucleotide 483 as set forth in SEQ ID NO:174 (HUMGCRFC_T5 transcript).

Alignment of the new FCγRIII-A variant (HUMGCRFC_P4—SEQ ID NO:175) with the WT protein (GenBank Accession No. P08637; FC3A_HUMAN; SEQ ID NO:173) revealed that the new variant includes the first 107 amino acids as of the WT protein (GenBank Accession No. P08637) followed by a unique 19 amino acid sequence [PFPTMTSCSLFVKSDYLVT (SEQ ID NO:176), FIG. 104 ]. The new variant uncovered by the present invention lacks the immunoglobulin/major histocompatibility complex (IPR003006) and immunoglobulin subtype (IPR003599) domain of the WT protein and therefore is expected to be a secreted, soluble protein (i.e., extracellular).

Comparison Report Between HUMGCRFC_P4 (SEQ ID NO:175) and FC3A_HUMAN (SEQ ID NO:173)

1. An isolated chimeric polypeptide HUMGCRFC_P4 (SEQ ID NO:175), comprising a first amino acid sequence being at least 90% homologous to MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAY SPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEV HIG corresponding to amino acids 1-107 of FC3A_HUMAN (SEQ ID NO:173), which also corresponds to amino acids 1-107 of HUMGCRFC_P4 (SEQ ID NO:175), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least % homologous to a polypeptide having the sequence PFPTMTSCSLFVKSDYLVT (SEQ ID NO:176) corresponding to amino acids 108-126 of HUMGCRFC_P4 (SEQ ID NO:175), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMGCRFC_P4 (SEQ ID NO:175), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence PFPTMTSCSLFVKSDYLVT (SEQ ID NO:176) in HUMGCRFC_P4 (SEQ ID NO:175).

Clinical Applications of the New FORM-A Variants

Since the variants uncovered by the present inventors, HUMGCRFC_P3 (SEQ ID NO:171) and HUMGCRFC_P4 (SEQ ID NO:175), include the first 107 amino acids of the WT FCγRIII-A, but yet, are missing the transmembrane domain and the immunoglobulin/major histocompatibility complex and immunoglobulin subtype domains, they can be used as antagonists for the endogenous FCγRIII-A. Thus, the FCγRIII-A variants of the present invention, SEQ ID NO:171 and/or SEQ ID NO:175 can be used as an anti-inflammatory, antiallergic, anti-asthma, antianaemic, antithrombotic, antiarthritic agent as well as an immunological, a cytokine, and an immunosuppressant agent.

Thus, the present inventors uncovered a therapeutic agent which can be used to treat inflammation, allergy, asthma, anaemic, thrombosis, and/or arthritis. Such an agent is a polypeptide homologous to SEQ ID NO:171 or 175, and/or a polynucleotide homologous to SEQ ID NO:174 or 170.

While further reducing the present invention to practice, the new variants of the present invention (HUMGCRFC_P3—SEQ ID NO:171 and/or HUMGCRFC_P4—SEQ ID NO: 175), the polynucleotide encoding same (HUMGCRFC_T4 (SEQ ID NO:170 and/or HUMGCRFC_T5—SEQ ID NO:174) and/or the peptide(s) derived from such variants (SEQ ID NO:172 and/or 176) can be used as diagnostic markers for allergy reactions, anaemia, rheumatoid arthritis, asthma, inflammation, lupus erythematosus, thrombocytopenia, thrombocytopenic purpura, large granular lymphocyte proliferative disease, and/or susceptibility to allograft failure (e.g., kidney allograft failure). Diagnosis according to this aspect of the present invention is effected using immunological assays [e.g., Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence, and the like using an antibody directed against the HUMGCRFC_P3 and/or HUMGCRFC_P4 (SEQ ID NO:171 and/or 175, respectively), or by nucleic acid techniques (NAT) such as RT-PCR, Northern Blot, in situ hybridization, in situ RT-PCR.

Splice Variant of Tumor Necrosis Factor Receptor Superfamily Member 3 Precursor

Lymphotoxin-β receptor (LT-βR) is a member of the tumor necrosis factor receptor (TNFR) superfamily and is expressed on the surface of most of cell types, including cells of epithelial and myeloid lineages but not on T and B lymphocytes. LT-βR can specifically bind two ligands: the membrane form of lymphotoxin, LT-α1/β2, which is uniquely expressed on activated lymphoid cells and LIGHT, a member of the TNF superfamily, which is induced on the cell surface during T cell activation. LT-βR has been speculated to play an essential role in the development of lymphoid organs. Thus, LT-β knock-out mice exhibit impaired lymph node development and loss of splenic architecture. In addition, LT-βR deficient mice were found to lack Peyer's patches, colon-associated lymphoid tissues and all lymph nodes. Moreover, stimulation of LT-βR on certain cell lines by LT-α1/β2 or anti-LTβR antibodies was found to induce cell death, chemokine secretion, and activation of nuclear factor κB (NFκB), suggesting an important biological function for LT-βR in mature individuals.

Like other members of the TNF receptor family, the cytoplasmic domain (CD) of LT-βR does not include consensus sequences characteristic of enzymatic activity. Thus, signaling is thought to be mediated by the proteins interacting with LT-βR such as the two serine/threonine protein kinases, p50 and p80 and the two members of the TNF receptor-associated factor (TRAF) family, TRAF3 and TRAF5, which specifically associate with the LT-βR(CD). Further study has indicated that TRAF3 plays an important role in mediating LT-βR-induced apoptosis, whereas TRAF5 involves in the activation of NFκB. On the other hand, several members of the TNFR superfamily (such as TNFRI, Fas, DR3, DR4, and DR5) contain a common motif, the death domain, in their cytoplasmic region that initiate the activation of caspase cascades to execute apoptosis. LT-βR(CD) does not contain a death domain, but signaling through LT-βR can also induce apoptosis. It was shown that the cytoplasmic domain of TNFRI can self-associate through its death domain, therefore overexpression of TNFRI or of the cytoplasmic domain thereof can induce receptor clustering, a crucial step for subsequent intracellular signaling. Despite the absence of the death domain, the LT-βR(CD) is capable of self-association. Thus, overexpression of LT-βR is sufficient to trigger apoptosis without the need for ligand conjugation (Tamada et al. 2002. The Journal of Clinical Investigation. Vol. 109, No. 4, Pp. 549-557; Shao et al. 2003. Eur. J. Immunol. 33:1736-1743; Ettinger et al. 1996. Proc. Natl. Acad. Sci. USA. Vol. 93, Pp. 13102-13107; Wu et al. 1999. The Journal of Biological Chemistry. Vol. 274, No. 17, Pp. 11868-11873; Hehlgans et al. 2002. Cancer Research 62:4034-4040).

Clinical Application

It has been shown that signaling through LT-βR induced cell death in some human adenocarcinoma tumor lines (HT-29 and WiDr) in the presence of IFN-γ. Combined in vivo treatment of human adenocarcinoma cells (WiDr), which form solid tumors in immunocompromised mice, with an agonistic anti-LT-βR antibody and human IFN-γ resulted in tumor growth arrest. Contrary to these findings, it has been shown that activation of LT-βR on fibrosarcoma tumor cells is necessary for angiogenesis and solid tumor growth. Prevention of LT-α1/β2-LT-βR signaling, by the release of LTβR-Fc from the tumor cells, inhibited tumor angiogenesis and neovascularization, and resulted in tumor growth arrest in mice. In addition, LT-βR activation on the tumor cells induced enhanced release of MIP-2, an angiogenic CXC chemokine. Thus, the interaction of activated LT-α1/β2-carrying lymphocytes with LT-βR-expressing tumor cells can initiate a novel pro-angiogenic pathway, leading to organized tumor tissue development. In addition to its modulation of tumor growth, LT-βR was shown to be involved in immune regulation. In vivo blockade of LIGHT and LTα1/β2 by administration of soluble lymphotoxin β receptor-Ig (LTβR-Ig) inhibited the cytotoxic T lymphocyte (CTL) response to host antigenic disparities and ameliorated lethal graft-versus-host disease (GVHD) in a B6 to BDF1 mouse model. In addition, it has been shown that treatment of rodents with the fusion protein, LT-βR-Ig, prevents the development of autoimmune diseases as insulitis and uveitis.

TNR3-LT-βR Splice Variant T2 Structure

A brief description is now provided of TNR3-LT-βR splice variants according to the present invention. TNR3 splice variant transcript — 2 (HUMTNFRRP_T2—SEQ ID NO:177; HUMTNFRRP_P2—SEQ ID NO:178). The protein coordinates on the transcript start from nucleotide 261 and end at nucleotide 1025 as set forth in SEQ ID NO:177.

TNR3 splice variant T2 results from alternative splicing of the TNR3 gene, introducing a novel exon, named exon 6a, between exons 6 and 7, leading to an insertion of a stop codon and the generation of a truncated protein. TNR3 splice variant T2 encodes a 255 amino acids long protein (HUMTNFRRP_P2—SEQ ID NO:178) which contains the N-terminal signal sequence (residues 1-30), the complete extracellular domain of TNR3, including all the TNFR CYS repeats, and a unique sequence of 33 amino acids [EPALSKGVENLQALLYQAATGSSEASFPTLSPL (SEQ ID NO:179), FIG. 105 ] at the C-terminus of the protein. However, as can see using the alignment with the WT sequence (TNR3_HUMAN; SEQ ID NO:129), the new variant uncovered by the present invention lacks the transmembrane domain (amino acids 228-248 of SEQ ID NO:129) and therefore is expected to be a secreted, soluble and extracellular protein.

Comparison Report Between HUMTNFRRP_P2 (SEQ ID NO:178) and TNR3_HUMAN (SEQ ID NO:129)

1. An isolated chimeric polypeptide HUMTNFRRP_P2 (SEQ ID NO:178), comprising a first amino acid sequence being at least 90% homologous to MLLPWATSAPGLAWGPLVLGLFGLLAASQPQAVPPYASENQTCRDQEKEYY EPQHRICCSRCPPGTYVSAKCSRIRDTVCATCAENSYNEHWNYLTICQLCRPC DPVMGLEEIAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTEAEL KDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCENQGLVEAAPGTAQSD TTCKNPLEPLPPEMS corresponding to amino acids 1-222 of TNR3_HUMAN (SEQ ID NO:129), which also corresponds to amino acids 1-222 of HUMTNFRRP_P2 (SEQ ID NO:178), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence EPALSKGVENLQALLYQAATGSSEASFPTLSPL (SEQ ID NO:179) corresponding to amino acids 223-255 of HUMTNFRRP_P2 (SEQ ID NO:178), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMTNFRRP_P2 (SEQ ID NO:178), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence EPALSKGVENLQALLYQAATGSSEASFPTLSPL (SEQ ID NO:179) in HUMTNFRRP_P2 (SEQ ID NO:178).

TNR3-LT-βR Splice Variant T18 Structure

TNR3 splice variant transcript — 18 (HUMTNFRRP_T18—SEQ ID NO:180; HUMTNFRRP_P2—SEQ ID NO:178). The protein coordinates on the transcript start from nucleotide 261 and end at nucleotide 1025 as set forth in SEQ ID NO:180. The protein encoded by this transcript (SEQ ID NO:180) is identical to the protein encoded by HUMTNFRRP_T2—SEQ ID NO:177, and is referred to herein as HUMTNFRRP_P2—SEQ ID NO:178.

The Therapeutic Potential of TNR3-LT-βR Splice Variants T2 and T18

TNR3 splice variants T2 and T18, each encode a soluble receptor which contains all four TNFR CYS repeats of the WT or known protein (TNR3_HUMAN; SEQ ID NO:129), but yet is a soluble, secreted protein. It can inhibit TNR3 signaling by competing on the ligand with the membrane-bound receptor, thus preventing LT-α1/β2 from binding to the cell surface receptor and activating it. A soluble form of TNR3 was shown already to bind LT-α1/β2 in vitro. Blocking LTαβ/TNR3 interactions was shown in vivo by administration of TNR3-Fc fusion protein or by the creation of mice which constitutively express a soluble murine TNR3-human IgG1 (Fc) transgene. Blocking TNR3 signaling could have important therapeutic potential for the treatment of cancer, graft-vs-host disease and autoimmune diseases, such as rheumatoid arthritis, Crohn's disease, insulitis and uveitis.

Thus, the TNR3 HUMTNFRRP_P2 (SEQ ID NO:178) of the present invention, the polynucleotide encoding same (TNR3 splice variants T2 and T18, SEQ ID NOs:177 and 180, respectively) and/or the unique peptide derived from the TNR3 HUMTNFRRP_P2 variant of the present invention (SEQ ID NO:179) can serve as a TNFR3 antagonist.

The present inventors uncovered a therapeutic agent which can be used to: (i) inhibit or prevent the binding of LT-α1/β2 to its TNR3 receptor in vivo or ex vivo, (ii) prevent tumor growth (e.g., solid tumor, such as fibrisarcoma) by preventing the activation of LT-βR, (iii) prevent and/or treat graft-versus-host disease (GVHD) by inhibition of the cytotoxic T lymphocyte (CTL) response, (iv) prevent and/or treat autoimmune diseases (e.g., insulitis, uveitis), (v) prevent and/or treat Crohn's disease, rheumatoid arthritis. Such an agent is a polypeptide homologous to HUMTNFRRP_P2 (SEQ ID NO:178), and/or a polynucleotide homologous to SEQ ID NO: 177 or 180 and/or a peptide homologous to SEQ ID NO:179). It will be appreciated that the polypeptide, polynucleotide and/or peptide used according to this aspect of the present can be administered or provided per se, or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

While further reducing the present invention to practice, the present inventor have uncovered that the TNR3 variant of the present invention is overexpressed in various cancers (e.g., stomach tumor) and therefore can be used as a diagnostic marker for such pathologies.

Diagnosis according to this aspect of the present invention is effected using immunological assays [e.g., Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence, and the like using an antibody directed against the HUMTNFRRP_P2 variant (SEQ ID NO:178), or by nucleic acid techniques (NAT) such as RT-PCR, Northern Blot, in situ hybridization, in situ RT-PCR.

Splice Variant of Granulocyte Colony Stimulating Factor Receptor Precursor (G-CSF-R)

The granulocyte colony stimulating factor receptor precursor (G-CSF-R) (CD114 antigen, GCSR_HUMAN; GenBank Accession No. Q99062) is a type I membrane protein which involves in cell adhesion, signal transduction, and defense response.

Clinical Applications

G-CSF-R has been implicated in anaemia, various cancers such as, breast cancer, leukaemia, acute myelogenous, leukaemia, lymphoma, melanoma, sarcoma, chemotherapy-induced injury, bone marrow, bone marrow leucopenia, bone marrow neutropenia, immunodeficiency, and infections.

The G-CSF-R is overexpressed in amniotic cells and placenta and thus can be used as a marker for cell proliferation in these tissues and as a marker for pathological de-differentiation of these tissues.

Splice Variant HSGCSFR2_T14 (SEQ ID NO:181) Encodes a New Secreted Form of the G-CSF-R, HSGCSFR2_P11 (SEQ ID NO:182)

The present inventors have uncovered a new G-CSF-R variant [HSGCSFR2_P11—SEQ ID NO:182; HSGCSFR2_T14—SEQ ID NO:181]. The protein coordinates on the transcript start from nucleotide 551 and end at nucleotide 2320 as set forth in SEQ ID NO:181 (HSGCSFR2_T14 transcript).

Alignment of the new G-CSF-R variant HSGCSFR2_P11—SEQ ID NO:182) with the WT protein (GenBank Accession No. Q99062; GCSR_HUMAN; SEQ ID NO:183) revealed that the new variant includes the first 525 amino acids as of the WT protein (GenBank Accession No. Q99062) followed by a unique 65 amino acid sequence [GLSTIRPLSRILSSLSQGSAWSPAIRSIGNIAFLPYFQPWRGSLPIPW LTLDPYSWTEIRCWDRN (SEQ ID NO:184), FIG. 106 ]. The new variant uncovered by the present invention lacks the fibronectin type-III domain (IPR003961; amino acids 527-618 in WT; SEQ ID NO:183) and the transmembrane domain (amino acids 628-650 of GenBank Accession No. Q99062; SEQ ID NO:183) of the WT protein and therefore is expected to be a secreted, soluble protein (i.e., extracellular).

Comparison Report Between HSGCSFR2_P11 and GCSR_HUMAN

1. An isolated chimeric polypeptide HSGCSFR2_P11 (SEQ ID NO:182), comprising a first amino acid sequence being at least 90% homologous to MARLGNCSLTWAALIILLLPGSLEECGHISVSAPIVHLGDPITASCIIKQNCSHL DPEPQILWRLGAELQPGGRQQRLSDGTQESIITLPHLNHTQAFLSCCLNWGNS LQILDQVELRAGYPPAIPHNLSCLMNLTTSSLICQWEPGPETHLPTSFTLKSFKS RGNCQTQGDSILDCVPKDGQSHCCIPRKHLLLYQNMGIWVQAENALGTSMSP QLCLDPMDVVKLEPPMLRTMDPSPEAAPPQAGCLQLCWEPWQPGLHINQKC ELRHKPQRGEASWALVGPLPLEALQYELCGLLPATAYTLQIRCIRWPLPGHW SDWSPSLELRTTERAPTVRLDTWWRQRQLDPRTVQLFWKPVPLEEDSGRIQG YVVSWRPSGQAGAILPLCNTTELSCTFHLPSEAQEVALVAYNSAGTSRPTPVV FSESRGPALTRLHAMARDPHSLWVGWEPPNPWPQGYVIEWGLGPPSASNSN KTWRMEQNGRATGFLLKENIRPFQLYEIIVTPLYQDTMGPSQHVYAYSQEM corresponding to amino acids 1-525 of GCSR_HUMAN (SEQ ID NO:183), which also corresponds to amino acids 1-525 of HSGCSFR2_P11 (SEQ ID NO:182), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence GLSTIRPLSRILSSLSQGSAWSPAIRSIGNIAFLPYFQPWRGSLPIPWLTLDPYSW TEIRCWDRN (SEQ ID NO:184) corresponding to amino acids 526-590 of HSGCSFR2_P11 (SEQ ID NO:182), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HSGCSFR2_P11 (SEQ ID NO:182), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GLSTIRPLSRILSSLSQGSAWSPAIRSIGNIAFLPYFQPWRGSLPIPWLTLDPYSW TEIRCWDRN (SEQ ID NO:184) in HSGCSFR2_P11 (SEQ ID NO:182).

Splice Variant HSGCSFR2_T8 (SEQ ID NO:185) Encodes a New Secreted Form of the G-CSF-R, HSGCSFR2_P7 (SEQ ID NO:186)

The present inventors have uncovered a new G-CSF-R variant [HSGCSFR2_P7—SEQ ID NO:186; HSGCSFR2_T8—SEQ ID NO:185]. The protein coordinates on the transcript start from nucleotide 551 and end at nucleotide 2281 as set forth in SEQ ID NO:185 (HSGCSFR2_T8 transcript).

Alignment of the new G-CSF-R variant (HSGCSFR2_P7—SEQ ID NO:186) with the WT protein (GenBank Accession No. Q99062; SEQ ID NO:183) revealed that the new variant includes the first 574 amino acids as of the WT protein (GenBank Accession No. Q99062) and is missing 66 amino acid sequence [SAILNASSRGFVLHGLEPASLYHIHLMAASQAGATNSTVLTLMTLTPEGSELH IILGLFGLLLLLT (SEQ ID NO:187), FIG. 107 ]. The new variant uncovered by the present invention exhibits a truncated fibronectin type-III domain (IPR003961; amino acids 527-618 in WT; GenBank Accession No. Q99062) and lacks the transmembrane domain of the WT protein (amino acids 628-650 of GenBank Accession No. Q99062) and therefore is expected to be a secreted, soluble protein (i.e., extracellular).

Comparison Report Between HSGCSFR2_P7 and GCSR_HUMAN

1. An isolated chimeric polypeptide HSGCSFR2_P7, comprising a first amino acid sequence being at least 90% homologous to MARLGNCSLTWAALIILLLPGSLEECGHISVSAPIVHLGDPITASCIIKQNCSHL DPEPQILWRLGAELQPGGRQQRLSDGTQESIITLPHLNHTQAFLSCCLNWGNS LQILDQVELRAGYPPAIPHNLSCLMNLTTSSLICQWEPGPETHLPTSFTLKSFKS RGNCQTQGDSILDCVPKDGQSHCCIPRKHLLLYQNMGIWVQAENALGTSMSP QLCLDPMDVVKLEPPMLRTMDPSPEAAPPQAGCLQLCWEPWQPGLHINQKC ELRHKPQRGEASWALVGPLPLEALQYELCGLLPATAYTLQIRCIRWPLPGHW SDWSPSLELRTTERAPTVRLDTWWRQRQLDPRTVQLFWKPVPLEEDSGRIQG YVVSWRPSGQAGAILPLCNTTELSCTFHLPSEAQEVALVAYNSAGTSRPTPVV FSESRGPALTRLHAMARDPHSLWVGWEPPNPWPQGYVIEWGLGPPSASNSN KTWRMEQNGRATGFLLKENIRPFQLYEIIVTPLYQDTMGPSQHVYAYSQEMA PSHAPELHLKHIGKTWAQLEWVPEPPELGKSPLTHYTIFWTNAQNQSF corresponding to amino acids 1-574 of GCSR_HUMAN (SEQ ID NO:183), which also corresponds to amino acids 1-574 of HSGCSFR2_P7 (SEQ ID NO:186), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence CLC corresponding to amino acids 575-577 of HSGCSFR2_P7 (SEQ ID NO:186), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

Splice Variant HSGCSFR2_T9 (SEQ ID NO:188) Encodes a New Secreted Form of the G-CSF-R, HSGCSFR2_P8 (SEQ ID NO:189)

The present inventors have uncovered a new G-CSF-R variant [HSGCSFR2_P8—SEQ ID NO:189; HSGCSFR2_T9—SEQ ID NO:188]. The protein coordinates on the transcript start from nucleotide 551 and end at nucleotide 2365 as set forth in SEQ ID NO:188 (HSGCSFR2_T9 transcript).

Alignment of the new G-CSF-R variant (HSGCSFR2_P8—SEQ ID NO:189) with the WT protein (GenBank Accession No. Q99062; SEQ ID NO:183) revealed that the new variant includes the first 574 amino acids as of the WT protein (GenBank Accession No. Q99062), followed by 31 amino acid sequence [CESILSSPTAPEGLEGGAQLPRRQLYHPGLC (SEQ ID NO:190), FIG. 108 ]. The new variant uncovered by the present invention exhibits a truncated fibronectin type-III domain (IPR003961; amino acids 527-618 in WT; GenBank Accession No. Q99062) and lacks the transmembrane domain of the WT protein (amino acids 628-650 of GenBank Accession No. Q99062) and therefore is expected to be a secreted, soluble protein (i.e., extracellular).

Comparison Report Between HSGCSFR2_P8 and GCSR_HUMAN

1. An isolated chimeric polypeptide HSGCSFR2_P8 (SEQ ID NO:189), comprising a first amino acid sequence being at least 90% homologous to MARLGNCSLTWAALIILLLPGSLEECGHISVSAPIVHLGDPITASCIIKQNCSHL DPEPQILWRLGAELQPGGRQQRLSDGTQESIITLPHLNHTQAFLSCCLNWGNS LQILDQVELRAGYPPAIPHNLSCLMNLTTSSLICQWEPGPETHLPTSFTLKSFKS RGNCQTQGDSILDCVPKDGQSHCCIPRKHLLLYQNMGIWVQAENALGTSMSP QLCLDPMDVVKLEPPMLRTMDPSPEAAPPQAGCLQLCWEPWQPGLHINQKC ELRHKPQRGEASWALVGPLPLEALQYELCGLLPATAYTLQIRCIRWPLPGHW SDWSPSLELRTTERAPTVRLDTWWRQRQLDPRTVQLFWKPVPLEEDSGRIQG YVVSWRPSGQAGAILPLCNTTELSCTFHLPSEAQEVALVAYNSAGTSRPTPVV FSESRGPALTRLHAMARDPHSLWVGWEPPNPWPQGYVIEWGLGPPSASNSN KTWRMEQNGRATGFLLKENIRPFQLYEIIVTPLYQDTMGPSQHVYAYSQEMA PSHAPELHLKHIGKTWAQLEWVPEPPELGKSPLTHYTIFWTNAQNQSF corresponding to amino acids 1-574 of GCSR_HUMAN (SEQ ID NO:183), which also corresponds to amino acids 1-574 of HSGCSFR2_P8 (SEQ ID NO:189), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence CESILSSPTAPEGLEGGAQLPRRQLYHPGLC (SEQ ID NO:190) corresponding to amino acids 575-605 of HSGCSFR2_P8 (SEQ ID NO:189), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HSGCSFR2_P8 (SEQ ID NO:189), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence CESILSSPTAPEGLEGGAQLPRRQLYHPGLC (SEQ ID NO:190) in HSGCSFR2_P8 (SEQ ID NO:189).

The expected pharmacological activity of the soluble G-CSF-R variants of the present invention (HSGCSFR2_P11, HSGCSFR2_P7 and HSGCSFR2_P8) are agonists of the granulocyte stimulating factor.

These results suggest the use of the new G-CSF-R variants of the present invention [HSGCSFR2_P11 (SEQ ID NO:182), HSGCSFR2_P7 (SEQ ID NO:186), and/or HSGCSFR2_P8 (SEQ ID NO:189)], the polynucleotides encoding same [HSGCSFR2_T14 (SEQ ID NO:181), HSGCSFR2_T8 (SEQ ID NO:185) and/or HSGCSFR2_T9 (SEQ ID NO:188)] and/or the peptides derived from the HSGCSFR2_P11 variant (SEQ ID NO:184) and/or the HSGCSFR2_P8 (SEQ ID NO:190) as a diagnostic marker for amniotic and/or placental cell proliferation or de-differentiation, as well as various tumors. Diagnosis according to this aspect of the present invention is effected using immunological assays [e.g., Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence, and the like using an antibody directed against the G-CSF-R variant (HSGCSFR2_P11—SEQ ID NO:182], nucleic acid techniques (NAT) such as RT-PCR, Northern Blot, in situ hybridization, in situ RT-PCR.

Moreover, G-CSF-R is implicated in various hematological and immunological conditions. Thus, the present inventors have uncovered that the new soluble form of G-CSF-R which can be used as an antianaemic, anticancer, immunomodulator, anti-infective, immunostimulant, radio or chemoprotective, anti-AIDS therapeutic agent.

It will be appreciated that such an agent can be administered or provided to an individual in need thereof per se or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

Splice Variant of E-Selectin Precursor

E-selectin (CD62E, ELAM-1) is a member of the selectins (CD62) family which also includes L-selectin (CD62L, LECAM-1, LAM-1) and P-selectin (CD62P, GMP140, PADGEM). E-selectin participates in recruiting leukocytes to sites of inflammation. Its expression is restricted in most cases to venules in site of acute and chronic inflammation and it was found to be expressed on cytokine-(TNFα, IL-1) activated endothelial cells. Furthermore, it is rapidly (within 4 hours) expressed on postcapillary venules in animal models of inflammation, supporting a role for this protein in the initial stages of the inflammatory response such as neutrophil recruitment. However, it seems that E-selectin expression is necessary but insufficient for T cell emigration into skin. Furthermore, E-selectin has some functional redundancy with P-selectin as in a model of DTH (T cell-mediated delayed type hypersensitivity) in E-selectin null mice P-selectin can compensate for the inflammatory response. Although upregulated upon stimulation by inflammatory cytokines, constitutive low levels of E-selectin expression appear to play a role in normal leukocyte trafficking.

Structurally, E-selectin contains an N-terminal C-type lectin domain (also designated carbohydrate-recognition domain, CRD), followed by an epidermal growth factor (EGF)-like domain, six short consensus repeats (SCRs, also designated sushi), a transmembrane domain, and a short cytoplasmic tail. The lectin domain of E-selectin is required for carbohydrate binding. However, mutagenesis studies have shown that although the c-lectin and EGF-like domains are the minimal functional unit, the presence of the six SCRs yields the most potent molecule in terms of binding of the E-selectin to cell surface and in inhibition of adhesion (Li et al., 1994).

Several cell types interact with E-selectin-expressing endothelium including myeloid cells, neutrophils, monocytes and certain subsets of lymphocytes (CD4, CD8 and memory). This interaction occurs via the recognition of carbohydrated, usually sialyl Lewis x (SLex)-bearing selectin ligands, including PSGL-1 (P-selectin glycoprotein ligand-1), L-selectin and CLA (cutaneous lymphocyte associated antigen).

Clinical Applications

Chronic inflammation that results from unregulated leukocyte interaction with the endothelium is reported in ischemia-reperfusion, rheumatoid arthritis, asthma, atherosclerosis, and multiple sclerosis. Additionally, E-selectin over expression and/or CLA+ T cell infiltrates have been associated with a number of chronic skin diseases such as atopic dermatitis, psoriasis, allergic contact dermatitis, cutaneous T cell lymphoma and other diseases (Rossiter et al., 1997). The chronic inflammatory response associated with overexpression of selectins is due, at least in part, to increased cytokine production by extravasated T cells that have encountered antigen and became activated Inhibition of extravasation by blocking selectins could prevent the initial accumulation in tissue, thereby preventing the subsequent production of cytokines. Interestingly, studies employing selectin knock out mice have shown that E-selectin null mice lacks a pathogenic phenotype (Frenette and Wagner, 1997).

E-selectin interaction with carbohydrates (mainly sialyl Lewis a but also x) have also been implicated in formation of metastases of several cancer cells. High amounts of sialyl Lewis (a) are present in human adenocarcinomas of the colon, pancreas and stomach. There are several lines of evidence showing that sialyl Lewis (a) is responsible for the adhesion of human cancer cells to endothelium. E-selectin present on endothelial cells mediates these interactions. Thus, E-selectin antagonists might block E-selectin/cancer cell contact and reduce the metastatic potential of the cancer cells (Ugorski and Laskowska, 2002). Additionally, E-selectin expression was shown to be involved in lymphocyte recruitment to the skin of patients with GvHD (Lange et al., 1995).

Elevated serum levels of soluble E-selectin could serve as a diagnostic marker of chronic inflammatory diseases and cancer as high levels of soluble E-selectin have been described in patients suffering from type II diabetes (Matsumoto et al., 2002), rheumatoid arthritis (Klimiuk et al., 2002), asthma (Hamzaoui et al., 2001), lupus, sepsis (Egerer et al., 2000), and colorectal and breast cancer (Ito et al., 2001, Matsuura et al., 1997). Furthermore, suppression of experimental lupus nephritis by elevated expression of soluble E-selectin in transgenic mice was reported (Takahashi et al., 2002), indicating that soluble E-selectin could also serve as a therapeutic protein in inflammation-associated diseases, even if high levels of the soluble molecules are present in the serum of these patients.

Splice Variant Structure

The present inventors uncovered a novel splice variant of E-selectin (HUMELAM1A_P2—SEQ ID NO:65 and HUMELAM1A_T1—SEQ ID NO:66; FIGS. 37 b and a, respectively). The splice variant HUMELAM1A_T1 was obtained by the alternative splicing of the E-selectin gene resulting in extension of exon 4 leading to an insertion of a stop codon and the generation of a truncated protein ( FIGS. 38-40 ). This splice variant encodes 189 amino acids long protein (SEQ ID NO:65), which contains 176 amino acids of the wild type sequence, and a unique sequence of 13 amino acids (SKSGSCLFLHLRW; SEQ ID NO:67). It encompasses the C-type lectin domain, the EGF-like domain, but lacks the six SCR (sushi) domains, the TM and the cytoplasmic domain. The variant retains the potential glycosylation sites relevant to the domains it encompasses.

Comparison Report Between HUMELAM1A_P2 (SEQ ID NO:65) and LEM2_HUMAN (SEQ ID NO:139)

1. An isolated chimeric polypeptide HUMELAM1A_P2 (SEQ ID NO:65), comprising a first amino acid sequence being at least 90% homologous to MIASQFLSALTLVLLIKESGAWSYNTSTEAMTYDEASAYCQQRYTHLVAIQN KEEIEYLNSILSYSPSYYWIGIRKVNNVWVWVGTQKPLTEEAKNWAPGEPNN RQKDEDCVEIYIKREKDVGMWNDERCSKKKLALCYTAACTNTSCSGHGECV ETINNYTCKCDPGFSGLKCEQ corresponding to amino acids 1-176 of LEM2_HUMAN (SEQ ID NO:139), which also corresponds to amino acids 1-176 of HUMELAM1A_P2 (SEQ ID NO:65), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence SKSGSCLFLHLRW (SEQ ID NO:67) corresponding to amino acids 177-189 of HUMELAM1A_P2 (SEQ ID NO:65), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of HUMELAM1A_P2 (SEQ ID NO:65), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SKSGSCLFLHLRW (SEQ ID NO:67) in HUMELAM1A_P2 (SEQ ID NO:65).

Therapeutic Application of the Splice Variant

E-selectin splice variant T1 contains the lectin-like domain and the EGF-like domain. As it lacks the transmembrane domain, it is predicted to be a soluble protein. Mutagenesis studies have shown that the lectin-like domain and the EGF-like domains contain the sites important for ligand binding. Thus, this variant is predicted to serve as an antagonist for E-selectin activity. However, as described above, the presence of the SCR domains elevate the activity of E-selectin constructs. Furthermore, a work by Lo and colleagues have shown that recombinant soluble E-selectin (comprised of the C-lectin, EGF-like and six SCR domains) serves as a chemoattractant for PMN and that immobilization of the recombinant E-selectin result in integrin activation. Taken together, these data demonstrate that the E-selectin variant T1 will be a very weak antagonist for leukocyte-endothelium interaction and might simultaneously serve a weak adhesion molecule if immobilized to the matrix.

Both agonist and antagonist for E-selectin are described in the pharma project. Most antagonists are antibodies and some of them are chemical or synthetic nucleic acid based molecules, which are designed to treat chronic inflammatory diseases and allergies, as well as cancer. The agonist is reported to be on preclinical status for treatment of ischemia.

Thus, the E-selectin variant of the present invention, the polypeptide, HUMELAM1A_P2 (SEQ ID NO:65), the polynucleotide encoding same, HUMELAM1A_T1 (SEQ ID NO:66) and/or the peptide derived from the splice variant of the present invention (SEQ ID NO:67) is an antagonist for leukocyte-endothelium interaction and thereby can be used as a therapeutic agent to treat various disorders such as chronic inflammation (e.g., ischemia-reperfusion, rheumatoid arthritis, asthma, atherosclerosis, and multiple sclerosis), chronic skin diseases (such as atopic dermatitis, psoriasis, allergic contact dermatitis, cutaneous T cell lymphoma).

According to another aspect of the present invention, the E-selectin variant of the present invention, the polypetide, HUMELAM1A_P2 (SEQ ID NO:65), the polynucleotide encoding same, HUMELAM1A_T1 (SEQ ID NO:66) and/or the peptide derived from the splice variant of the present invention (SEQ ID NO:67) is an antagonist for the E-selectin interaction with carbohydrates and thus can be used as a therapeutic agent to prevent the formation of metastases of several cancer cells (e.g., adenocarcinomas of the colon, pancreas and stomach) and/or prevent the adhesion of human cancer cells to endothelium.

Without being bound to any theory, the E-selectin variant of the present invention, the polypetide, HUMELAM1A_P2 (SEQ ID NO:65), the polynucleotide encoding same, HUMELAM1A_T1 (SEQ ID NO:66) and/or the peptide derived from the splice variant of the present invention (SEQ ID NO:67) is an E-selectin agonist and thus can be used as a therapeutic agent for the treatment of ischemia.

It will be appreciated that such agent(s) can be administered or provided to an individual in need thereof per se or as part of a pharmaceutical composition with a pharmaceutical acceptable carrier (e.g., PEG and liposomes).

According to another aspect of the present invention, the E-selectin variant of the present invention, the polypetide, HUMELAM1A_P2 (SEQ ID NO:65), the polynucleotide encoding same, HUMELAM1A_T1 (SEQ ID NO:66) and/or the peptide derived from the splice variant of the present invention (SEQ ID NO:67) are diagnostic marker(s) for chronic inflammatory diseases (e.g., type II diabetes, rheumatoid arthritis, asthma, lupus, sepsis) and cancers (e.g., colorectal and breast cancer).

Diagnosis according to this aspect of the present invention is effected using immunological assays [e.g., Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence, and the like using an antibody directed against the HUMELAM1A_P2 (SEQ ID NO:65) variant, or by nucleic acid techniques (NAT) such as RT-PCR, Northern Blot, in situ hybridization, in situ RT-PCR.

Splice Variant of Integrin Alpha-L Precursor

Integrin αL (CD11a) forms a heterodimer with integrin β2 (CD18) to generate LFA-1. LFA-1 functions as an intercellular adhesion receptor and as a co-stimulatory and signaling molecule. It mediates the interaction between cytotoxic T cells and their targets, and functions in T and B cells as a mitogen, and in B cells as an inducer of B cell aggregation and Ig production. It interacts with ICAM-1 (CD54), ICAM-2 (CD102), ICAM-3 (CD54), ICAM-4 (Landsteiner-Weiner antigen), or ICAM-5 (telencephalin). High levels of LFA-1 are constitutively expressed on lymphocytes and monocytes, and lower levels on neutrophils. The ability of LFA-1 to interact with its ligands and transmit signals across the plasma membrane is regulated by a variety of extracellular and intracellular factors. These include agents that alter integrin affinity or avidity such as divalent cations Mg 2+ , Ca 2+ , Mn 2+ , the state of cell activation, intracellular Ca 2+ release, activation of PKC, and ligands themselves. The regulation of LFA-1 activity by these factors avoid inappropriate adhesion while these cells are circulating in blood or migrating in tissues.

Structurally, the extracellular region of LFA-1 contains seven homologues tandem repeats (I-VII) that fold as a β propeller. Domains V-VII contain putative divalent cation binding sites also designated EF-hands. Between repeats II and III there is insertion of ˜200 amino acids, known as the I domain, which is considered to be functionally implicated in binding of ligands and cations and contains a discontinued MIDAS. The extracellular portion of integrin αL contains 12 potential N-glycosylation sites and seven disulfide bonds.

Mouse knock out model of integrin αL displays defects in T cell proliferation and cytotoxicity and in tumor rejection but lymphocyte homing, leukocyte extravasation and thymic maturation and selection do not seem to be defected (Cabanas and Sanchez-Madrid, 1999).

Clinical Applications

Inhibition of CD11a, LFA-1, or its ligand have been shown to be a useful target for therapy of several inflammatory situations associated with accumulation of leukocytes leading to clot formation or cytotoxicity. Monoclonal Abs to CD11a, ICAM-1, and CD18 were comparably effective in a rabbit model of myocardial reperfusion injury and reduced infract size by 40-50%, but only if administered well before reperfusion. Reducing cytotoxic T cell activity by mAbs to CD11a/CD18, in combination with standard immunosuppressive therapy, improve the survival of bone marrow transplants in children but not in adults. In a mouse model, anti CD11a mAbs increased the survival of allogenic tumor grafts. LFA-1 might also be involved in cancer metastasis as its expression on hematological tumor cells have been shown to alter metastatic capacity and growth (Mazzone and Richevuti, 1995).

Anti CD11a mAb is a registered drug for treatment of psoriasis (Cabanas and Sanchez-Madrid, 1999 J Biol Regul Homeost Agents 13: 126-129; Mazzone and Richevuti, 1995 Hematologic a 80:161-175; Ihanus et al., 2003 Eur J Biochem 270: 1710-1723; McDowell et al., 1998 JBC 273: 27396-27403; Binnerts et al., 1996 JBC 271: 9962-9968.)

Splice Variant Structure

The present inventors uncovered a novel splice variant of integrin αL (T83460_T11—SEQ ID NO:81; T83460_P8—SEQ ID NO:80; FIGS. 49 a and b , respectively). The T11 splice variant (T83460_T11) obtained by the alternative splicing of the integrin αL gene result in extension of exon 18 leading to an insertion of a stop codon and the generation of a truncated protein ( FIGS. 50-51 ). This splice variant encodes 750 amino acids long protein (SEQ ID NO:80), which contains 745 amino acids of the wild type sequence, and 5 unique amino acids (VRRDG; SEQ ID NO:82). It encompasses the FG-GAPS I and VII, while lacking the TM and the cytoplasmic domain. It contains seven out of 12 potential N-glycosilation sites and four out of seven disulfide bonds.

Comparison Report Between T83460_P8 (SEQ ID NO:80) and ITAL_HUMAN_V1 (SEQ ID NO:653)

1. An isolated chimeric polypeptide T83460_P8 (SEQ ID NO:80), comprising a first amino acid sequence being at least 90% homologous to MKDSCITVMAMALLSGFFFFAPASSYNLDVRGARSFSPPRAGRHFGYRVLQV GNGVIVGAPGEGNSTGSLYQCQSGTGHCLPVTLRGSNYTSKYLGMTLATDPT DGSILACDPGLSRTCDQNTYLSGLCYLFRQNLQGPMLQGRPGFQECIKGNVD LVFLFDGSMSLQPDEFQKILDFMKDVMKKLSNTSYQFAAVQFSTSYKTEFDFS DYVKRKDPDALLKHVKHMLLLTNTFGAINYVATEVFREELGARPDATKVLIII TDGEATDSGNIDAAKDIIRYIIGIGKHFQTKESQETLHKFASKPASEFVKILDTF EKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSRGHAVVGAVGA KDWAGGFLDLKADLQDDTFIGNEPLTPEVRAGYLGYTVTWLPSRQKTSLLAS GAPRYQHMGRVLLFQEPQGGGHWSQVQTIHGTQIGSYFGGELCGVDVDQDG ETELLLIGAPLFYGEQRGGRVFIYQRRQLGFEEVSELQGDPGYPLGRFGEAITA LTDINGDGLVDVAVGAPLEEQGAVYIFNGRHGGLSPQPSQRIEGTQVLSGIQW FGRSIHGVKDLEGDGLADVAVGAESQMIVLSSRPVVDMVTLMSFSPAEIPVH EVECSYSTSNKMKEGVNITICFQIKSLIPQFQGRLVANLTYTLQLDGHRTRRRG LFPGGRHELRRNIAVTTSMSCTDFSFHFPVCVQDLISPINVSLNFSLWEEEGTP RDQRA corresponding to amino acids 1-745 of ITAL_HUMAN_V1 (SEQ ID NO:653), which also corresponds to amino acids 1-745 of T83460_P8 (SEQ ID NO:80), and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRRDG (SEQ ID NO:82) corresponding to amino acids 746-750 of T83460_P8 (SEQ ID NO:80), wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of T83460_P8 (SEQ ID NO:80), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRRDG (SEQ ID NO:82) in T83460_P8 (SEQ ID NO:80).

Comparison Report Between T83460_P8 (SEQ ID NO:80) and ITAL_HUMAN (SEQ ID NO:142)

1. An isolated chimeric polypeptide T83460_P8 (SEQ ID NO:80), comprising a first amino acid sequence being at least 90% homologous to MKDSCITVMAMALLSGFFFFAPASSYNLDVRGARSFSPPRAGRHFGYRVLQV GNGVIVGAPGEGNSTGSLYQCQSGTGHCLPVTLRGSNYTSKYLGMTLATDPT DGSILACDPGLSRTCDQNTYLSGLCYLFRQNLQGPMLQGRPGFQECIKGNVD LVFLFDGSMSLQPDEFQKILDFMKDVMKKLSNTSYQFAAVQFSTSYKTEFDFS DYVKRKDPDALLKHVKHMLLLTNTFGAINYVATEVFREELGARPDATKVLIII TDGEATDSGNIDAAKDIIRYIIGIGKHFQTKESQETLHKFASKPASEFVKILDTF EKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSRGHAVVGAVGA KDWAGGFLDLKADLQDDTFIGNEPLTPEVRAGYLGYTVTWLPSRQKTSLLAS GAPRYQHMGRVLLFQEPQGGGHWSQVQTIHGTQIGSYFGGELCGVDVDQDG ETELLLIGAPLFYGEQRGGRVFIYQRRQLGFEEVSELQGDPGYPLGRFGEAITA LTDINGDGLVDVAVGAPLEEQGAVYIFNGRHGGLSPQPSQRIEGTQVLSGIQW FGRSIHGVKDLEGDGLADVAVGAESQMIVLSSRPVVDMVTLMSFSPAEIPVH EVECSYSTSNKMKEGVNITICFQIKSL corresponding to amino acids 1-659 of ITAL_HUMAN (SEQ ID NO:142), which also corresponds to amino acids 1-659 of T83460_P8 (SEQ ID NO:80), a bridging amino acid I corresponding to amino acid 660 of T83460_P8 (SEQ ID NO:80), a second amino acid sequence being at least 90% homologous to PQFQGRLVANLTYTLQLDGHRTRRRGLFPGGRHELRRNIAVTTSMSCTDFSFH FPVCVQDLISPINVSLNFSLWEEEGTPRDQRA corresponding to amino acids 661-745 of ITAL_HUMAN (SEQ ID NO:142), which also corresponds to amino acids 661-745 of T83460_P8 (SEQ ID NO:80), and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRRDG (SEQ ID NO:82) corresponding to amino acids 746-750 of T83460_P8 (SEQ ID NO:80), wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.

2. An isolated polypeptide for a tail of T83460_P8 (SEQ ID NO:80), comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRRDG (SEQ ID NO:82) in T83460_P8 (SEQ ID NO:80).

Therapeutic Applications of the Variant

Immobilized recombinant CD11a I-domain fused to GST was shown to bind ICAM-4 positive cells (Ihanus et al., 2003). In addition, McDowell et al. have shown that recombinant soluble form of CD11a I domain inhibits the binding of Mg 2+ activated T cells (high affinity binding) but not phorbol ester-activated T cells (low affinity) to ICAM-1. The conclusion drown from this study suggests that the I domain is involved in a conformational change which is necessary for binding activity and that soluble I domain (or its fragments) interferes with interdomain alterations or intersubunit associations and thereby prevent changes in the quarternary structure of LFA-1. Accordingly, T11, which encompass the whole I domain as well as the MIDAS, is predicted to function as an antagonist of LFA-1 activity and to inhibit leukocyte adhesion to LFA-1 substrates. As the I domains for different ligands are overlapping but not identical (Ihanus et al., 2003), the antagonistic activity of T11 will probably be non-ligand specific.

Thus, a soluble αL variant, i.e., a polypeptide homologous to SEQ ID NO:80, a polynucleotide homologous to SEQ ID NO:81, and/or a peptide homologous to SEQ ID NO:82 is an antagonist of LFA-1 activity.

Thus, the present inventors uncovered a therapeutic agent which can be used to: (i) inhibit leukocyte adhesion to LFA-1 substrates, (ii) prevent the interaction between cytotoxic T cell and their targets, (iii) inhibit leukocyte accumulation associated with inflammation. Such an agent can be used (i) in the treatment of reperfusion injury, (ii) as an immunosuppressant (by reducing cytotoxic T cell activity) in transplantations, (iii) in the treatment of psoriasis. Such an agent is a polypeptide homologous to SEQ ID NO:80, and/or a polynucleotide homologous to SEQ ID NO:81 and/or a peptide homologous to SEQ ID NO:82.

Splice Variant of Macrophage Inflammatory Protein-2-Beta Precursor

The macrophage inflammatory protein-2-beta precursor (MIP2-beta; CXCL3; Growth regulated protein gamma; GRO-gamma; GRO3; GROG; SCYB3) is a secreted protein exhibiting chemokine activity for neutrophils. MIP2-β may have a role in inflammation and immune response and exerts its effects on endothelial cells in an autocrine fashion. In vitro studies showed that a processed polypeptide having amino acids 5-73 of the WT protein (GenBank Accession NO. P19876) exhibits a 5-fold higher chemotactic activity for neutrophilic granulocytes, suggesting a functional role for this part of the protein.

MIP2-β is overexpressed in lung and can be used as a marker for proliferation of this tissue or as a marker for pathological de-differentiation of this tissue or tissue damage.

Splice Variant T11329_T1 (SEQ ID NO:191) Encodes a New Secreted Form of the MIP2-β, T11329_P2 (SEQ ID NO:192)

The present inventors have uncovered a new MIP2-β variant [T11329_T1-SEQ ID NO:191; T11329_P2—SEQ ID NO:1921. The protein coordinates on the transcript start from nucleotide 84 and end at nucleotide 614 as set forth in SEQ ID NO:191 (T11329_T1 transcript).

Alignment of the new MIP2-β variant (T11329_P2—SEQ ID NO:192) with the WT protein (GenBank Accession No. P19876; SEQ ID NO:193) revealed that the new variant includes additional 103 amino acids [MHKKGSPILGSHTARVAGTSPPALPLLAQLPDASAEPHGPRHALRRPQQS PAPAGGAAAPAPGGRQPARSRWVPAPWGPRAGRGWGGRPAPTAPLNQRVY SSL (SEQ ID NO:88), FIG. 109 ] followed by amino a